Human collagenase inhibitor, recombinant vector system for using same and recombinant DNA method for the manufacture of same

ABSTRACT

A portable DNA sequence is disclosed which is capable of directing intracellular production of metalloproteinanse inhibitors. Vectors containing this portable DNA sequence are also set forth, including the vector pUC9-F5/237P10 (ATCC Accession No. 53003). A recombinant-DNA method for the microbial production of a metalloproteinase inhibitor, which method incorporates at least one of the portable DNA sequences and the vectors disclosed herein.

BACKGROUND OF THE INVENTION

[0001] This is a continuation-in-part application of U.S. patentapplication Ser. No. 699,181, filed Feb. 5, 1985. Endogenous proteolyticenzymes serve to degrade invading organisms, antigen-antibody complexesand certain tissue proteins which are no longer necessary or useful tothe organism. In a normally functioning organism, proteolytic enzymesare produced in a limited quantity and are regulated in part throughspecific inhibitors.

[0002] Metalloproteinases are enzymes present in the body which areoften involved in the degradation of connective tissue. While someconnective tissue degradation is necessary for normal functioning of anorganism, an excess of connective tissue degradation occurs in severaldisease states and is believed to be attributable, at least in part, toexcess metalloproteinase. It is believed that metalloproteinases are atleast implicated in periodontal disease, corneal and skin ulcers,rheumatoid arthritis and the spread of cancerous solid tumors.

[0003] These diseases generally occur in areas of the body which containa high proportion of collagen, a particular form of connective tissue.An examination of patients with these diseases of connective tissue hasrevealed an excessive breakdown of the various components of connectivetissues, including collagen proteoglycans and elastin. Therefore, it hasbeen deduced that an excessive concentration of a particularmetalloproteinase, for example collagenase, proteoglyconuse, gelatinase,and certain elastases, may cause or exacerbate the connective tissuedestruction associated with the aforementioned diseases.

[0004] In the normal state, the body possesses metalloproteinaseinhibitors which bind to metalloproteinases to effectively prevent theseenzymes from acting on their connective tissue substrates. Specifically,in a healthy organism, metalloproteinase inhibitors are present inconcentrations sufficient to interact with metalloproteinases to anextent which allows sufficient quantities of metalloproteinase to remainactive while binding the excess metalloproteinase so that the connectivetissue damage seen in the various diseases does not occur.

[0005] It is postulated that one immediate cause of the connectivetissue destruction present in the foregoing disease states is animbalance in the relative metalloproteinase/metalloproteinase inhibitorconcentrations. In these situations, either due to an excessive amountof active metalloproteinase or a deficiency in the amount of activemetalloproteinase inhibitor, the excess metalloproteinase is believed tocause the connective tissue degradation responsible for causing orexacerbating the disease. It is postulated that, by treating personswith connective tissue diseases with metalloproteinase inhibitors, thedegradative action of the excess metalloproteinase may be curtailed orhalted. Therefore, particular metalloproteinase inhibitors of specificinterest to the present inventors are collagenase inhibitors because itis believed that these inhibitors would be pharmaceutically useful inthe treatment or prevention of connective tissue diseases.

[0006] The existence of metalloproteinase and metalloproteinaseinhibitors has been discussed in the scientific literature. For example,Sellers et al., Biochemical And Biophysical Research Communications87:581-587 (1979), discusses isolation of rabbit bone collagenaseinhibitor. Collagenase inhibitor isolated from human skin fibroblasts isdiscussed in Stricklin and Welgus, J. B. C. 258:12252-12258 (1983) andWelgus and Stricklin, J. B. C. 258:12259-12264 A1983). The presence ofcollagenase inhibitors in naturally-occurring body fluids is furtherdiscussed in Murphy et al., Biochem. J. 195:167-170 (1981) and Cawstonet al., Arthritis and Rheumatism, 27:285 (1984). In addition,metalloproteinase inhibitors are discussed by Reynolds et al. inCellular Interactions, Dingle and Gordon, eds., (1981). Although thesearticles characterize particular, isolated metalloproteinase inhibitorsand discuss, to some extent, the role or potential role ofmetalloproteinases in connective tissue disease treatment and speculateon the ability of metalloproteinase inhibitors to counteract thisdestruction, none of these researchers had previously been able toisolate a portable DNA sequence capable of directing intracellularproduction of metalloproteinase inhibitors or to create arecombinant-DNA method for the production of these inhibitors.

[0007] Surprisingly, the present inventors have discovered a portableDNA sequence capable of directing the recombinant-DNA synthesis ofmetalloproteinase inhibitors. These metalloproteinase inhibitors arebiologically equivalent to those isolated from human skin fibroblastcultures. The metalloproteinase inhibitors of the present invention,prepared by the recombinant-DNA methods set forth herein, will enableincreased research into prevention and treatment ofmetalloproteinase-induced connective tissue diseases. In addition, themetalloproteinase inhibitors of the present invention are useful inneutralizing metalloproleinases, including the excess metalloproteinaseassociated with disease states. Therefore, it is believed that a curefor these diseases will be developed which will embody, as an activeingredient, the metalloproteinase inhibitors of the present invention.Furthermore, the metalloproteinase inhibitors of the present inventionare capable of interacting with their metalloproteinase targets in amanner which allows the development of diagnostic tests for degradativeconnective tissue diseases using the newly discovered inhibitors.

[0008] The recombinant metalloproteinase inhibitors discussed hereininteract stoichiometrically (i.e., in a 1:1 ratio) with theirmetalloproteinase targets. In addition, these metalloproteinaseinhibitors are heat resistant, acid stable, glycosylated, and exhibit ahigh isoelectric point.

SUMMARY OF THE INVENTION

[0009] The present invention relates to metalloproteinase inhibitors anda recombinant-DNA method of producing the same and to portable DNAsequences capable of directing intracellular production of themetalloproteinase inhibitors. Particularly, the present inventionrelates to a collagenase inhibitor, a recombinant-DNA method forproducing the same and to portable DNA sequences for use in therecombinant method. The present invention also relates to a series ofvectors containing these portable DNA sequences.

[0010] One object of the present invention is to provide ametalloproteinase inhibitor, which can be produced in sufficientquantities and purities to provide economical pharmaceuticalcompositions which possess metalloproteinase inhibitor activity.

[0011] An additional object of the present invention is to provide arecombinant-DNA method for the production of these metalloproteinaseinhibitors. The recombinant metalloproteinase inhibitors produced bythis method are biologically equivalent to the metalloproteinaseinhibitor isolable from human skin fibroblast cultures.

[0012] To facilitate the recombinant-DNA synthesis of thesemetalloproteinase inhibitors, it is a further object of the presentinvention to provide portable DNA sequences capable of directingintracellular production of metalloproteinase inhibitors. It is also anobject of the present invention to provide cloning vectors containingthese portable sequences. These vectors are capable of being used inrecombinant systems to produce pharmaceutically useful quantities ofmetalloproteinase inhibitors.

[0013] Additional objects and advantages of the invention will be setforth in part in the description which follows, and in part will beobvious from the description or may be learned from practice of theinvention. The objects and advantages may be realized and attained bymeans of the instrumentalities and combinations particularly pointed outin the appended claims.

[0014] To achieve the objects and in accordance with the purposes of thepresent invention, metalloproteinase inhibitors are set forth, which arecapable of stoichiometric reaction with metalloproteinases. Thesemetalloproteinase inhibitors are remarkably heat resistant, acid stable,glycosylated, and exhibit a high isoelectric point. Furthermore, thesemetalloproteinase inhibitors are biologically equivalent to thoseinhibitors isolated from human skin fibroblast cultures.

[0015] To further achieve the objects and in accordance with thepurposes of the present invention, as embodied and broadly describedherein, portable DNA sequences coding for metalloproteinase inhibitorsare provided. These sequences comprise nucleotide sequences capable ofdirecting intracellular production of metalloproteinase inhibitors. Theportable sequences may be either synthetic sequences or restrictionfragments (“natural” DNA sequences). In a preferred embodiment, aportable DNA sequence is isolated from a human fibroblast cDNA libraryand is capable of directing intracellular production of a collagenaseinhibitor which is biologically equivalent to that inhibitor which isisolable from a human skin fibroblast culture.

[0016] The coding strand of a first preferred DNA sequence which hasbeen discovered has the following nucleotide sequence:         10        20         30         40         50         60 GTTGTTGCTGTGGCTGATAG CCCCAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACAG         70        80         90        100        110        120 ACGGCCTTCTGCAATTCCGA CCTCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAGTC        130       140        150        160        170        180 AACCAGACCACCTTATACCA GCGTTATGAG ATCAAGATGA CCAAGATGTA TAAAGGGTTC        190       200        210        220        230        240 CAAGCCTTAGGGGATGCCGC TGACATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGTC        250       260        270        280        290        300 TGCGGATACTTCCACAGGTC CCACAACCGC AGCGAGGAGT TTCTCATTGC TGGAAAACTG        310       320        330        340        350        360 CAGGATGGACTCTTGCACAT CACTACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAGC        370       380        390        400        410        420 TTAGCTCAGCGCCGGGGCTT CACCAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGTC        430       440        450        460        470        480 TTTCCCTGTTTATCCATCCC CTGCAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGAC        490       500        510        520        530        54 CAGCTCCTCCAAGGCTCTGA AAAGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCG        550       560        570        580        590        60 GAGCCAGGGCTGTGCACCTG GCAGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGG        610       620        630        640        650        66 GTGGAAGCTGAAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGAC        670       680        690        700 ATGAAATAAA GAGTTACCAC CCAGCAAAAAAAAAAAGGAA TTC

[0017] The nucleotides represented by the foregoing abbreviations areset forth in the Detailed Description of the Preferred Embodiments.

[0018] A second preferred DNA sequence has been discovered which has anadditional nucleotide sequence 5′ to the initiator sequence. Thissequence, which contains as the eighty-second throughfour-hundred-thirty-second nucleotides nucleotides 1 through 351 of thefirst preferred sequence set forth above, has the following nucleotidesequence:         10         20         30         40         50        6 GGCCATCGCC GCAGATCCAG CGCCCAGAGA GACACCAGAG AACCCACCATGGCCCCCTT         70         80         90        100        110       1 GACCCCTGGC TTCTGCATCC TGTTGTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCA       130        140        150        160        170        1TGTGTCCCAC CCCACCCACA GACGGCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCA       190        200        210        220        230        2TTCGTGGGGA CACCAGAAGT CAACCAGACC ACCTTATACC AGCGTTATGA GATCAAGA       250        260        270        280        290        3ACCAAGATGT ATAAAGGGTT CCAAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCT       310        320        330        340        350        3ACCCCCGCCA TGGAGAGTGT CTGCGGATAC TTCCACAGGT CCCACAACCG CAGCGAGG       370        380        390        400        410        42TTTCTCATTG CTGGAAAACT GCAGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGT       430 GCTCCCTGGA AC

[0019] A third preferred DNA sequence which incorporates the 5′ regionof the second preferred sequence and the 3′ sequence of the firstpreferred sequence, has the following nucleotide sequence:         10        20         30         40         50         6 GGCCATCGCCGCAGATCCAG CGCCCAGAGA GACACCAGAG AACCCACCAT GGCCCCCTT         70        80         90        100        110       12 GACCCCTGGCTTCTGCATCC TGTTGTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCAC        130       140        150        160        170        18 TGTGTCCCACCCCACCCACA GACGGCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCAA        190       200        210        220        230        24 TTCGTGGGGACACCAGAAGT CAACCAGACC ACCTTATACC AGCGTTATGA GATCAAGAT        250       260        270        280        290        30 ACCAAGATGTATAAAGGGTT CCAAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCTA        310       320        330        340        350        36 ACCCCCGCCATGGAGAGTGT CTGCGGATAC TTCCACAGGT CCCACAACCG CAGCGAGGA        370       380        390        400        410        42 TTTCTCATTGCTGGAAAACT GCAGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGT        430       440        450        460        470        48 GCTCCCTGGAACAGCCTGAG CTTAGCTCAG CGCCGGGGCT TCACCAAGAC CTACACTGT        490       500        510        520        530        54 GGCTGTGAGGAATGCACAGT GTTTCCCTGT TTATCCATCC CCTGCAAACT GCAGAGTGG        550       560        570        580        590        60 ACTCATTGCTTGTGGACGGA CCAGCTCCTC CAAGGCTCTG AAAAGGGCTT CCAGTCCCC        610       620        630        640        650        66 CACCTTGCCTGCCTGCCTCG GGAGCCAGGG CTGTGCACCT GGCAGTCCCT GCGGTCCCA        670       680        690        700        710        72 ATAGCCTGAATCCTGCCCGG AGTGGAAGCT GAAGCCTGCA CAGTGTCCAC CCTGTTCCC        730       740        750        760        770        780 CTCCCATCTTTCTTCCGGAC AATGAAATAA AGAGTTACCA CCCAGCAAAA AAAAAAAGGA To facilitateidentification and isolation of natural DNA

[0020] sequences for use in the present invention, the inventors havedeveloped a human skin fibroblast cDNA library. This library containsthe genetic information capable of directing a cell to synthesize themetalloproteinase inhibitors of the present invention. Other natural DNAsequences which may be used in the recombinant DNA methods set forthherein may be isolated from human genomic libraries.

[0021] Additionally, portable DNA sequences useful in the processes ofthe present invention may be synthetically created. These synthetic DNAsequences may be prepared by polynucleotide synthesis and sequencingtechniques known to those of ordinary skill in the art.

[0022] Additionally, to achieve the objects and in accordance with thepurposes of the present invention, a recombinant-DNA method is disclosedwhich results in microbial manufacture of the instant metalloproteinaseinhibitors using the portable DNA sequences referred to above. Thisrecombinant DNA method comprises:

[0023] (a) preparation of a portable DNA sequence capable of directing ahost microorganism to produce a protein having metalloproteinaseinhibitor activity, preferably collagenase inhibitor activity;

[0024] (b) cloning the portable DNA sequence into a vector capable ofbeing transferred into and replicating in a host microorganism, suchvector containing operational elements for the portable DNA sequence;

[0025] (c) transferring the vector containing the portable DNA sequenceand operational elements into a host microorganism capable of expressingthe metalloproteinase inhibitor protein;

[0026] (d) culturing the host microorganism under conditions appropriatefor amplification of the vector and expression of the inhibitor; and

[0027] (e) in either order:

[0028] (i) harvesting the inhibitor; and

[0029] (ii) causing the inhibitor to assume an active, tertiarystructure whereby it possesses metalloproteinase inhibitor activity.

[0030] To further accomplish the objects and in further accord with thepurposes of the present invention, a series of cloning vectors areprovided comprising at least one of the portable DNA sequences discussedabove. In particular, plasmid pUC9-F5/237P10 is disclosed.

[0031] It is understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

[0032] The accompanying drawing, which is incorporated in andconstitutes a part of this specification, illustrates one embodiment ofthe invention and, together with the description, serves to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 is a partial restriction map of the plasmid pUC9-F5/237P10.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the drawing and thefollowing examples, serve to explain the principles of the invention.

[0035] As noted above, the present invention relates in part to portableDNA sequences capable of directing intracellular production ofmetalloproteinase inhibitors in a variety of host microorganisms.“Portable DNA sequence” in this context is intended to refer either to asynthetically-produced nucleotide sequence or to a restriction fragmentof a naturally occuring DNA sequence. For purposes of thisspecification, “metalloproteinase inhibitor” is intended to mean theprimary structure of the protein as defined by the codons present in thedeoxyribonucleic acid sequence which directs intracellular production ofthe amine acid sequence, and which may or may not includepost-translational modifications. It is contemplated that suchpost-translational modifications include, for example, glycosylation. Itis further intended that the term “metalloproteinase inhibitor” refer toeither the form of the protein as would be excreted from a microorganismor the methionyl-metalloproteinase inhibitor as it may be present inmicroorganisms from which it was not excreted.

[0036] In a preferred embodiment, the portable DNA sequences are capableof directing intracellular production of collagenase inhibitors. In aparticularly preferred embodiment, the portable DNA sequences arecapable of directing intracellular production of a collagenase inhibitorbiologically equivalent to that previously isolated from human skinfibroblast cultures. By “biologically equivalent”, as used herein in thespecification and claims, it is meant that an inhibitor, produced usinga portable DNA sequence of the present invention, is capable ofpreventing collagenase-induced tissue damage of the same type, but notnecessarily to the same degree, as a native human collagenase inhibitor,specifically that native human collagenase inhibitor isolable from humanskin fibroblast cell cultures.

[0037] A first preferred portable DNA sequence of the present inventionhas a nucleotide sequence as follows:         10         20         30        40         50         60 GTTGTTGCTG TGGCTGATAG CCCCAGCAGGGCCTGCACCT GTGTCCCACC CCACCCACAG         70         80         90       100        110        120 ACGGCCTTCT GCAATTCCGA CCTCGTCATCAGGGCCAAGT TCGTGGGGAC ACCAGAAGTC        130        140        150       160        170        180 AACCAGACCA CCTTATACCA GCGTTATGAGATCAAGATGA CCAAGATGTA TAAAGGGTTC        190        200        210       220        230        240 CAAGCCTTAG GGGATGCCGC TGACATCCGGTTCGTCTACA CCCCCGCCAT GGAGAGTGTC        250        260        270       280        290        300 TGCGGATACT TCCACAGGTC CCACAACCGCAGCGAGGAGT TTCTCATTGC TGGAAAACTG        310        320        330       340        350        360 CAGGATGGAC TCTTGCACAT CACTACCTGCAGTTTCGTGG CTCCCTGGAA CAGCCTGAGC        370        380        390       400        410        420 TTAGCTCAGC GCCGGGGCTT CACCAAGACCTACACTGTTG GCTGTGAGGA ATGCACAGTG        430        440        450       460        470        480 TTTCCCTGTT TATCCATCCC CTGCAAACTGCAGAGTGGCA CTCATTGCTT GTGGACGGAC        490        500        510       520        530        540 CAGCTCCTCC AAGGCTCTGA AAAGGGCTTCCAGTCCCGTC ACCTTGCCTG CCTGCCTCGG        550        560        570       580        590        600 GAGCCAGGGC TGTGCACCTG GCAGTCCCTGCGGTCCCAGA TAGCCTGAAT CCTGCCCGGA        610        620        630       640        650        660 GTGGAAGCTG AAGCCTGCAC AGTGTCCACCCTGTTCCCAC TCCCATCTTT CTTCCGGACA        670        680        690       700 ATGAAATAAA GAGTTACCAC CCAGCAAAAA AAAAAAGGAA TTC

[0038] wherein the following nucleotides are represented by theabbreviations indicated below. Nucleotides Abbreviation Deoxyadenylicacid A Deoxyguanylic acid G Deoxycytidylic acid C Thymidylic acid T

[0039] A second preferred portable DNA sequence of the present inventionhas the following nucleotide sequence:         10         20         30        40         50         60 GGCCATCGCC GCAGATCCAG CGCCCAGAGAGACACCAGAG AACCCACCAT GGCCCCCTTT         70         80         90       100        110        120 GACCCCTGGC TTCTGCATCC TGTTGTTGCTGTGGCTGATA GCCCCAGCAG GGCCTGCACC        130        140        150       160        170        180 TGTGTCCCAC CCCACCCACA GACGGCCTTCTGCAATTCCG ACCTCGTCAT CAGGGCCAAG        190        200        210       220        230        240 TTCGTGGGGA CACCAGAAGT CAACCAGACCACCTTATACC AGCGTTATGA GATCAAGATG        250        260        270       280        290        300 ACCAAGATGT ATAAAGGGTT CCAAGCCTTAGGGGATGCCG CTGACATCCG GTTCGTCTAC        310        320        330       340        350        360 ACCCCCGCCA TGGAGAGTGT CTGCGGATACTTCCACAGGT CCCACAACCG CAGCGAGGAG        370        380        390       400        410        420 TTTCTCATTG CTGGAAAACT GCAGGATGGACTCTTGCACA TCACTACCTG CAGTTTCGTC        430 GCTCCCTGGA AC

[0040] In this second preferred sequence, an open reading frame existsfrom nucleotides 1 through 432. The first methionine of this readingframe is encoded by nucleotides by 49 through 51 and is the site oftranslation initiation. It should be noted that the amino acid sequenceprescribed nucleotides 49 through 114 is not found in the maturemetalloproteinase. It is believed that this sequence is the leaderpeptide of the human protein.

[0041] A third preferred portable DNA sequence has the mucleotidesequence.         10         20         30         40         50        60 GGCCATCGCC GCAGATCCAG CGCCCAGAGA GACACCAGAG AACCCACCATGGCCCCCTTT         70         80         90        100        110       120 GACCCCTGGC TTCTGCATCC TGTTGTTGCT GTGGCTGATA GCCCCAGCAGGGCCTGCACC        130        140        150        160        170       180 TGTGTCCCAC CCCACCCACA GACGGCCTTC TGCAATTCCG ACCTCGTCATCAGGGCCAAG        190        200        210        220        230       240 TTCGTGGGGA CACCAGAAGT CAACCAGACC ACCTTATACC AGCGTTATGAGATCAAGATG        250        260        270        280        290       300 ACCAAGATGT ATAAAGGGTT CCAAGCCTTA GGGGATGCCG CTGACATCCGGTTCGTCTAC        310        320        330        340        350       360 ACCCCCGCCA TGGAGAGTGT CTGCGGATAC TTCCACAGGT CCCACAACCGCAGCGAGGAG        370        380        390        400        410       420 TTTCTCATTG CTGGAAAACT GCAGGATGGA CTCTTGCACA TCACTACCTGCAGTTTCGTG        430        440        450        460        470       480 GCTCCCTGGA ACAGCCTGAG CTTAGCTCAG CGCCGGGGCT TCACCAAGACCTACACTGTT        490        500        510        520        530       540 GGCTGTGAGG AATGCACAGT GTTTCCCTGT TTATCCATCC CCTGCAAACTGCAGAGTGGC        550        560        570        580        590       600 ACTCATTGCT TGTGGACGGA CCAGCTCCTC CAAGGCTCTG AAAAGGGCTTCCAGTCCCGT        610        620        630        640        650       660 CACCTTGCCT GCCTGCCTCG GGAGCCAGGG CTGTGCACCT GGCAGTCCCTGCGGTCCCAG        670        680        690        700        710       720 ATAGCCTGAA TCCTGCCCGG AGTGGAAGCT GAAGCCTGCA CAGTGTCCACCCTGTTCCCA        730        740        750        760        770       780 CTCCCATCTT TCTTCCGGAC AATGAAATAA AGAGTTACCA CCCAGCAAAAAAAAAAAGGA

[0042] This third sequence contains the 5′ nontranslated region of thesecond preferred sequence and the 3′ region of the first preferredsequence. It is envisioned that this third preferred sequence is capableof directing intracellular production of a metalloproteinase analogousto a mature human collagenase inhibitor in a microbial or mammalianexpression system.

[0043] It must be borne in mind in the practice of the present inventionthat the alteration of some amino acids in a protein sequence may notaffect the fundamental properties of the protein. Therefore, it is alsocontemplated that other portable DN, sequences, both those capable ofdirecting intracellular production of identical amino acid sequences andthose capable of directing intracellular production of analogous aminoacid sequences which also possess metalloproteinase inhibitor activity,are included within the ambit of the present invention.

[0044] It is contemplated that some of these analogous amino acidsequences will be substantially homologous to native humanmetalloproteinase inhibitors while other amino acid sequences, capableof functioning as metalloproteinase inhibitors, will not exhibitsubstantial homology to native inhibitors. By “substantial homology”, asused herein, is meant a degree of homology to a native metalloproteinaseinhibitor in excess of 50%, preferably in excess of 60%, preferably inexcess of 80%. The percentage homology as discussed herein is calculatedas the percentage of amino acid residues found in the smaller of the twosequences that align with identical amino acid residues in the sequencebeing compared when four gaps in a length of 100 amino acids may beintroduced to assist in that alignment as set forth by Dayhoff, M. O. inAtlas of Protein Sequence and Structure Vol. 5, p. 124 (1972), NationalBiochemical Research Foundation, Washington, D.C., specificallyincorporated herein by reference.

[0045] As noted above, the portable DNA sequences of the presentinvention may be synthetically created. It is believed that the meansfor synthetic creation of these polynucleotide sequences are generallyknown to one of ordinary skill in the art, particularly in light of theteachings contained herein. As an example of the current state of theart relating to polynucleotide synthesis, one is directed to Matteucci,M. D. and Caruthers, M. H., in J. Am. Chem. Soc. 103: 3185 (1981) andBeaucage, S. L. and Caruthers, M. H. in Tetrahedron Lett. 22: 1859(1981), specifically incorporated herein by reference.

[0046] Additionally, the portable DNA sequence may be a fragment of anatural sequence, i.e., a fragment of a polynucleotide which occurred innature and which has been isolated and purified for the first time bythe present inventors. In one embodiment, the portable DNA sequence is arestriction fragment isolated from a cDNA library. In this preferredembodiment, the cDNA library is created from human skin fibroblasts.

[0047] In an alternative embodiment, the portable DNA sequence isisolated from a human genomic library. An example of such a libraryuseful in this embodiment is set forth in Lawn et al. Cell 15: 1157-1174(1978), specifically incorporated herein by reference.

[0048] As also noted above, the present invention relates to a series ofvectors, each containing at least one of the portable DN, sequencesdescribed herein. It is contemplated that additional copies of theportable DNA sequence may be included in a single vector to increase ahost microorganism's ability to produce large quantities of the desiredmetalloproteinase inhibitor.

[0049] In addition, the cloning vectors within the scope of the presentinvention may contain supplemental nucleotide sequences preceding orsubsequent to the portable DNA sequence. These supplemental sequencesare those that will not interfere with transcription of the portable DNAsequence and will, in some instances as set forth more fullyhereinbelow, enhance transcription, translation, or the ability of theprimary amino acid structure of the resultant metalloproteinaseinhibitor to assume an active, tertiary form.

[0050] A preferred vector of the present invention is set forth inFIG. 1. This vector, pUC9-F5/237P10, contains the preferred nucleotidesequence set forth above. Vector pUC9-F5/237P10 is present in theC600/pUC9-F5/237P10 cells on deposit in the American Type CultureCollection in Rockville, Md. under Accession No. 53003.

[0051] A preferred nucleotide sequence encoding the metalloproteinaseinhibitor is identified in FIG. 1 as region A. Plasmid pUC9-F5/237P10also contains supplemental nucleotide sequences preceding and subsequentto the preferred portable DNA sequence in region A. These supplementalsequences are identified as regions B and C, respectively.

[0052] In alternate preferred embodiments, either one or both of thepreceding or subsequent supplemental sequences may be removed from thevector of FIG. 1 by treatment of the vector with restrictionendonucleases appropriate for removal of the supplemental sequences. Thesupplemental sequence subsequent to the portable DNA sequence,identified in FIG. 1 as region C, may be removed by treatment of thevector with a suitable restriction endonuclease, preferably HgiAIfollowed by reconstruction of the 3′ end of region A using syntheticoligonucleotides and ligation of the vector with T-4 DNA ligase.Deletion of the supplemental sequence preceding the portable DNAsequence, identified as region B in FIG. 1, would be specificallyaccomplished by the method set forth in Example 2.

[0053] In preferred embodiments, cloning vectors containing and capableof expressing the portable DNA sequence of the present invention containvarious operational elements. These “operational elements,” as discussedherein, include at least one promoter, at least one Shine-Dalgarnosequence, at least one terminator codon. Preferably, these “operationalelements” also include at least one operator, at least one leadersequence, and for proteins to be exported from intracellular space, atleast one regulator and any other DNA sequences necessary or preferredfor appropriate transcription and subsequent translation of the vectorDNA.

[0054] Additional embodiments of the present invention are envisioned asemploying other known or currently undiscovered vectors which wouldcontain one or more of the portable DNA sequences described herein. Inparticular, it is preferred that these vectors have some or all of thefollowing characteristics: (1) possess a minimal number of host-organismsequences; (2) be stable in the desired host; (3) be capable of beingpresent in a high copy number in the desired host; (4) possess aregulatable promoter; (5) have at least one DNA sequence coding for aselectable trait present on a portion of the plasmid separate from thatwhere the portable DNA sequence will be inserted; and (6) b, integratedinto the vector.

[0055] The following, noninclusive, list of cloning vectors is believedto set forth vectors which can easily be altered to meet theabove-criteria and are therefore preferred for use in the presentinvention. Such alterations are easily performed by those of ordinaryskill in the art in light of the available literature and the teachingsherein. TABLE I HOST Vectors Comments E. coli pUC8 Many selectablereplicons pUC9 have been characterized. pBR322 Maniatis, T. et al.(1982), pGW7 Molecular Cloning: A placI^(q) Laboratory Manual, Cold pDP8Spring Harbor Laboratory. BACILLUS pUB110 Genetics and Biotechnology B.subtilis pSA0501 of Bacilli, Ganesan and B. amyloliquefaciens pSA2100Hoch, eds., 1984, Academic B. stearothermophilus pBD6 Press. pBD8 pT127PSEUDOMONAS RSF1010 Some vectors useful in P. aeruginosa Rms149 broadhost range of gram- P. putida pKT209 negative bacteria including RK2Xanthomonas and Agrobacteric pSa727 CLOSTRIDIUM pJU12 Shuttle plasmidsfor E. C. perfringens pJU7 coli and C. perfringens pJU10 constructionref. Squires, pJU16 C. et al. (1984) Journal pJU13 Bacteriol.159:465-471. SACCHAROMYCES YEp24 Botstein and Davis in S. cerevisiaeYIp5 Molecular Biology of the YRp17 Yeast Saccharomyces, Strathern,Jones, and Broach, eds., 1982, Cold Spring Harbor Laboratory.

[0056] It is to be understood that additional cloning vectors may nowexist or will be discovered which have the above-identified propertiesand are therefore suitable for use in the present invention. Thesevectors are also contemplated as being within the scope of the disclosedseries of cloning vectors into which the portable DNA sequences may beintroduced, along with any necessary operational elements, and whichaltered vector is then included within the scope of the presentinvention and would be capable of being used in the recombinant-DNAmethod set forth more fully below.

[0057] In addition to the above list, an E. coli vector system, as setforth in Example 2, is preferred in one embodiment as a cloning vector.Moreover, several vector plasmids which autonomously replicate in abroad range of Gram Negative bacteria are preferred for use as cloningvehicles in hosts of the genera Pseudomonas. These are described byTait, R. C., Close, T. J., Lundquist, R. C., Hagiya, M., Rodriguez, R.L., and Kado, C. I. in Biotechnology, May, 1983, pp. 269-275;Panopoulos, N. J. in Genetic Engineering in the Plant Sciences, PraegerPublishers, New York, N.Y., pp. 163-185, (1981); and Sakaguchi, K. inCurrent Topic in Microbiology and Immunology 96:31-45, (1982), each ofwhich is specifically incorporated herein by reference.

[0058] One particularly preferred construction employs the plasmidRSF1010 and derivatives thereof as described by Bagdasarian, M.,Bagdasarian, M. M., Coleman, S., and Timmis, K. N. in Plasmids ofMedical Environmental and Commercial Importance, Timmis, K. N. andPuhler, A. eds., Elsevier/North Holland Biomedical Press, (1979),specifically incorporated herein by reference. The advantages of RSF1010are that it is relatively small, high copy number plasmid which isreadily transformed into and stably maintained in both E. coli andPseudomonas species. In this system, it is preferred to use the Tacexpression system as described for Escherichia, since it appears thatthe E. coli trp promoter is readily recognized by Pseudomonas RNApolymerase as set forth by Sakaguchi, K. in Current Topics inMicrobiology and Immunology 96:31-45 (1982) and Gray, G. L., McKeown, K.A., Jones, A. J. S., Seeburg, P. H., and Heyneker, H. L. inBiotechnology February 1984, pp. 161-165, both of which are specificallyincorporated herein by reference. Transcriptional activity may befurther maximized by requiring the exchange of the promoter with, e.g.,an E. coli or P. aeruginosa trp promoter.

[0059] In a preferred embodiment, P. aeruginosa is transformed withvectors directing the synthesis of the metalloproteinase inhibitor aseither an intracellular product or as a product coupled to leadersequences that will effect its processing and export from the cell. Inthis embodiment, these leader sequences are preferably selected from thegroup consisting of beta-lactamase, OmpA protein, the naturallyoccurring human signal peptide, and that of carboxyeptidase G2 fromPseudomonas. Translation may be coupled to translation initiation forany of the E. coli proteins as described in Example 2, as well as toinitiation sites for any of the highly expressed proteins of the host tocause intracellular expression of the metalloproteinase inhibitor.

[0060] In those cases where restriction minus strains of a hostPseudomonas species are not available, transformation efficiency withplasmid constructs isolated from E. coli are poor. Therefore, passage ofthe Pseudomonas cloning vector through an r−m+ strain of another speciesprior to transformation of the desired host, as set forth inBagdasarian, M., et al., Plasmids of Medical. Environmental andCommercial Importance, pp. 411-422, Timmis and Puhler eds.,Elsevier/North Holland Biomedical Press (1979), specificallyincorporated herein by reference, is desired.

[0061] Furthermore, a preferred expression system in hosts of the generaBacillus involves using plasmid pUB110 as the cloning vehicle. As inother host vector systems, it is possible in Bacillus to express themetalloproteinase inhibitors of the present invention as either anintracellular or a secreted protein. The present embodiments includeboth systems. Shuttle vectors that replicate in both Bacillus and E.coli are available for constructing and testing various genes asdescribed by Dubnau, D., Gryczan, T., Contente, S., and Shivakumar, A.G. in Genetic Engineering, Vol. 2, Setlow and Hollander eds., PlenumPress, New York, N.Y., pp. 115-131, (1980), specifically incorporatedherein by reference. For the expression and secretion ofmetalloproteinase inhibitors from B. subtilis, the signal sequence ofalpha-amylase is preferably coupled to the coding region for themetalloproteinase inhibitor. For synthesis of intracellularmetalloproteinase inhibitor, the portable DNA sequence will betranslationally coupled to the ribosome binding site of thealpha-amylase leader sequence.

[0062] Transcription of either of these constructs is preferablydirected by the alpha-amylase promoter or a derivative thereof. Thisderivative contains the RNA polymerase recognition sequence of thenative alpha-amylase promoter but incorporates the lac operator regionas well. Similar hybrid promoters constructed from the penicillinasegene promoter and the lac operator have been shown to function inBacillus hosts in a regulatable fashion as set forth by Yansura, D. G.and Henner in Genetics and Biotechnology of Bacilli, Ganesan, A. T. andHoch, J. A., eds., Academic Press, pp. 249-263, (1984), specificallyincorporated by reference. The lacI gene of lacI^(q) would also beincluded to effect regulation.

[0063] One preferred construction for expression in Clostridium is inplasmid pJU12 described by Squires, C. H. et al in J. Bacteriol.159:465-471 (1984), specifically incorporated herein by reference,transformed into C. perfringens by the method of Heefner, D. L. et al.as described in J. Bacteriol. 159:460-464 (1984), specificallyincorporated herein by reference. Transcription is directed by thepromoter of the tetracycline resistance gene. Translation is coupled tothe Shine-Dalgarno sequences of this same tet^(r) gene in a mannerstrictly analogous to the procedures outlined above for vectors suitablefor use in other hosts.

[0064] Maintenance of foreign DNA introduced into yeast can be effectedin several ways (Botstein, D., and Davis, R. W., in The MolecularBiology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory,Strathern, Jones and Broach, eds., pp. 607-636 (1982). One preferredexpression system for use with host organisms of the genus Saccharomycesharbors the anticollagenase gene on the 2 micron plasmid. The advantagesof the 2 micron circle include relatively high copy number and stabilitywhen introduced into cir° strains. These vectors perferably incorporatethe replication origin and at least one antibiotic resistance markerfrom pBR322 to allow replication and selection in E. coli. In addition,the plasmid will preferably have 2 micron sequences and the yeast LEU2gene to serve the same purposes in LEU2 mutants of yeast.

[0065] The regulatable promoter from the yeast GALL gene will preferablybe adapted to direct transcription of the portable DNA sequence gene.Translation of the portable DNA sequence in yeast will be coupled to theleader sequence that directs the secretion of yeast alpha-factor. Thiswill cause formation of a fusion protein which will be processed inyeast and result in secretion of a metalloproteinase inhibitor.Alternatively, a methionyl-metalloproteinase inhibitor will betranslated for inclusion within the cell.

[0066] It is anticipated that translation of mRNA coding for themetalloproteinase inhibitor in yeast will be more efficient with thepreferred codon usage of yeast than with the sequence present inpUC8-Fic, as identified in Example 2, which has been tailored to theprokaryotic bias. For this reason, the portion of the 5′ end of theportable DNA sequence beginning at the TthlllI site i preferablyresynthesized. The new sequence favors the codons most frequently usedin yeast. This new sequence preferably has the following nucleotidesequence:            HgiAI 5′ GAT CCG TGC ACT TGT GTT CCA CCA CAC        GC ACG TGA ACA CAA GGT GGT GTG    CCA CAA ACT GCT TTC TGT AACTCT GAC C    GGT GTT TGA CGA AAG ACA TTG AGA CTG GA 3′

[0067] As will be seen from an examination of the individual cloningvectors and systems contained on the above list and description, variousoperational elements may be present in each c the preferred vectors ofthe present invention. It is contemplated any additional operationalelements which may be required may be added to these vectors usingmethods known to those of ordinary skill in the art, particularly inlight of the teachings herein.

[0068] In practice, it is possible to construct each of these vectors ina way that allows them to be easily isolated, assembled, andinterchanged. This facilitates assembly of numerous functional genesfrom combinations of these elements and the coding region of themetalloproteinase inhibitor. Further, many of these elements will beapplicable in more than one host.

[0069] At least one origin of replication recognized by the contemplatedhost microorganism, along with at least one selectable marker and atleast one promoter sequence capable of initiating transcription of theportable DNA sequence are contemplated as being included in thesevectors. It is additionally contemplated that the vectors, in certainpreferred embodiments, will contain DNA sequences capable of functioningas regulators (“operators”), and other DNA sequences capable of codingfor regulator proteins. In preferred vectors of this series, the vectorsadditionally contain ribosome binding sites, transcription terminatorsand leader sequences.

[0070] These regulators, in one embodiment, will serve to preventexpression of the portable DNA sequence in the presence of certainenvironmental conditions and, in the presence of other environmentalconditions, allow transcription and subsequent expression of the proteincoded for by the portable DNA sequence. In particular, it is preferredthat regulatory segments be inserted into the vector such thatexpression of the portable DNA sequence will not occur in the absenceof, for example, isopropylthio-^(β)-d-galactoside. In this situation,the transformed microorganisms containing the portable DNA may be grownto a desired density prior to initiation of the expression of themetalloproteinase inhibitor. In this embodiment, expression of thedesired protease inhibitor is induced by addition of a substance to themicrobial environment capable of causing expression of the DNA sequenceafter the desired density has been achieved.

[0071] Additionally, it is preferred that an appropriate secretoryleader sequence be present, either in the vector or at the 5′ end of theportable DNA sequence, the leader sequence being in a position whichallows the leader sequence to be immediately adjacent to the initialportion of the nucleotide sequence capable of directing expression ofthe protease inhibitor without any intervening transcription ortranslation termination signals. The presence of the leader sequence isdesired in part for one or more of the following reasons: 1) thepresence of the leader sequence may facilitate host processing of theinitial product to the mature recombinant metalloproteinase inhibitor;2) the presence of the leader sequence may facilitate purification ofthe recombinant metalloproteinase inhibitors, through directing themetalloproteinase inhibitor out of the cell cytoplasm; 3) the presenceof the leader sequence may affect the ability of the recombinantmetalloproteinase inhibitor to fold to its active structure throughdirecting the metalloproteinase inhibitor out of the cell cytoplasm.

[0072] In particular, the leader sequence may direct cleavage of theinitial translation product by a leader peptidase to remove the leadersequence and leave a polypeptide with the amino acid sequence which hasthe potential of metalloproteinase inhibitory activity. In some speciesof host microorganisms, the presence of the appropriate leader sequencewill allow transport of the completed protein into the periplasmicspace, as in the case of E. coli. In the case of certain yeasts andstrains of Bacillus and Pseudomonas, the appropriate leader sequencewill allow transport of the protein through the cell membrane and intothe extracellular medium. In this situation, the protein may be purifiedfrom extracellular protein.

[0073] Thirdly, in the case of some of the metalloproteinase inhibitorsprepared by the present invention, the presence of the leader sequencemay be necessary to locate the completed protein in an environment whereit may fold to assume its active structure, which structure possessesthe appropriate metalloproteinase activity.

[0074] Additional operational elements include, but are not limited to,ribosome-binding sites and other DNA sequences necessary for microbialexpression of foreign proteins. The operational elements as discussedherein can be routinely selected by those of ordinary skill in the artin light of prior literature and the teachings contained herein. Generalexamples of these operational elements are set forth in B. Lewin, Genes,Wiley & Sons, New York (1983), which is specifically incorporated hereinby reference. Various examples of suitable operational elements may befound on the vectors discussed above and may be elucidated throughreview of the publications discussing the basic characteristics of theaforementioned vectors.

[0075] In one preferred embodiment of the present invention, anadditional DNA sequence is located immediately preceding the portableDNA sequence which codes for the metalloproteinase inhibitor. Theadditional DNA sequence is capable of functioning as a translationalcoupler, i.e., it is a DNA sequence that encodes an RNA which serves toposition ribosomes immediately adjacent to the ribosome binding site ofthe metalloproteinase inhibitor RNA with which it is contiguous.

[0076] Upon synthesis and/or isolation of all necessary and desiredcomponent parts of the above-discussed cloning vectors, the vectors areassembled by methods generally known to those of ordinary skill in theart. Assembly of such vectors is believed to be within the duties andtasks performed by those with ordinary skill in the art and, as such, iscapable of being performed without undue experimentation. For examplesimilar DNA sequences have been ligated into appropriate cloningvectors, as set forth in Schoner et al., Proceedings of the NationalAcademy of Sciences U.S.A., 81:5403-5407 (1984), which is specificallyincorporated herein by reference.

[0077] In construction of the cloning vectors of the present invention,it should additionally be noted that multiple copies of the portable DNAsequence and its attendant operational elements may be inserted intoeach vector. In such an embodiment, the host organism would producegreater amounts per vector of the desired metalloproteinase inhibitor.The number of multiple copies of the DNA sequence which may be insertedinto the vector is limited only by the ability of the resultant vector,due to its size, to be transferred into and replicated and transcribedin an appropriate host microorganism.

[0078] Additionally, it is preferred that the cloning vector contain aselectable marker, such as a drug resistance marker or other markerwhich causes expression of a selectable trait by the host microorganism.In a particularly preferred embodiment of the present invention, thegene for ampicillin resistance is included in vector pUC9-F5/237P10.

[0079] Such a drug resistance or other selectable marker is intended inpart to facilitate in the selection of transformants. Additionally, thepresence of such a selectable marker on the cloning vector may be of usein keeping contaminating microorganisms from multiplying in the culturemedium. In This embodiment, such a pure culture of the transformed hostmicroorganisms would be obtained by culturing the microorganisms underconditions which require the induced phenotype for survival.

[0080] It is noted that, in preferred embodiment, it is also desirableto reconstruct the 3′ end of the coding region to allow assembly with 3′non-translated sequences. Included among these non-translated sequencesare those which stabilize the mRNA or enhance its transcription andthose that provide strong transcriptional termination signals which maystabilize the vector as they are identified by Gentz, R., Langner, A.,Chang, A. C. Y., Cohen, S. H., and Bujard, H. in Proc. Natl. Acad. Sci.USA 78:4936-4940 (1981), specifically incorporated herein by reference.

[0081] This invention also relates to a recombinant-DNA method for theproduction of metallproteinase inhibitors. Generally, this methodincludes:

[0082] (a) preparation of a portable DNA sequence capable of directing ahost microorganism to produce a protein having metalloproteinaseinhibitor activity;

[0083] (b) cloning the portable DNA sequence into a vector capable ofbeing transferred into and replicating in a host microorganism, suchvector containing operational elements for the portable DNA sequence;

[0084] (c) transferring the vector containing the portable DNA sequenceand operational elements into a host microorganism capable of expressingthe metalloproteinase inhibitor protein;

[0085] (d) culturing the host microorganism under conditions appropriatefor amplification of the vector and expression of the inhibitor; and

[0086] in either order:

[0087] (i) harvesting the inhibitor; and

[0088] (ii) causing the inhibitor to assume an active, tertiarystructure whereby it possesses metalloproteinase inhibitor activity.

[0089] In this method, the portable DNA sequences are those synthetic ornaturally-occurring polynucleotides described above. In a preferredembodiment of the present method, the portable DNA sequence has thenucleotide sequence as follows:         10         20         30        40         50         60 GTTGTTGCTG TGGCTGATAG CCCCAGCAGGGCCTGCACCT GTGTCCCACC CCACCCACAG         70         80         90       100        110        120 ACGGCCTTCT GCAATTCCGA CCTCGTCATCAGGGCCAAGT TCGTGGGGAC ACCAGAAGTC        130        140        150       160        170        180 AACCAGACCA CCTTATACCA GCGTTATGAGATCAAGATGA CCAAGATGTA TAAAGGGTTC        190        200        210       220        230        240 CAAGCCTTAG GGGATGCCGC TGACATCCGGTTCGTCTACA CCCCCGCCAT GGAGAGTGTC        250        260        270       280        290        300 TGCGGATACT TCCACAGGTC CCACAACCGCAGCGAGGAGT TTCTCATTGC TGGAAAACTG        310        320        330       340        350        360 CAGGATGGAC TCTTGCACAT CACTACCTGCAGTTTCGTGG CTCCCTGGAA CAGCCTGAGC        370        380        390       400        410        420 TTAGCTCAGC GCCGGGGCTT CACCAAGACCTACACTGTTG GCTGTGAGGA ATGCACAGTG        430        440        450       460        470        480 TTTCCCTGTT TATCCATCCC CTGCAAACTGCAGAGTGGCA CTCATTGCTT GTGGACGGAC        490        500        510       520        530        540 CAGCTCCTCC AAGGCTCTGA AAAGGGCTTCCAGTCCCGTC ACCTTGCCTG CCTGCCTCGG        550        560        570       580        590        600 GAGCCAGGGC TGTGCACCTG GCAGTCCCTGCGGTCCCAGA TAGCCTGAAT CCTGCCCGGA        610        620        630       640        650        660 GTGGAAGCTG AAGCCTGCAC AGTGTCCACCCTGTTCCCAC TCCCATCTTT CTTCCGGACA        670        680        690       700 ATGAAATAAA GAGTTACCAC CCAGCAAAAA AAAAAAGGAA TTC

[0090] The vectors contemplated as being useful in the present methodare those described above. In a preferred embodiment, the cloning vectorpUC9-F5/237P10 is used in the disclosed method.

[0091] The vector thus obtained is then transferred into the appropriatehost microorganism. It is believed that any microorganism having theability to take up exogenous DNA and express those genes and attendantoperational elements may be chosen. It is preferred that the hostmicroorganism be an anaerobe, facultative anaerobe or aerobe. Particularhosts which may be preferable for use in this method include yeasts andbacteria. Specific yeasts include those of the genus Saccharomyces, andespecially Saccharomyces cerevisiae.

[0092] Specific bacteria include those of the genera Bacillus andEscherichia and Pseudomonas. various other preferred hosts are set forthin Table I, supra. In other, alternatively preferred embodiments of thepresent invention, Bacillus subtilis, Escherichia coli or Pseudomonasaeruginosa are selected as the host microorganisms.

[0093] After a host organism has been chosen, the vector is transferredinto the host organism using methods generally known by those ofordinary skill in the art. Examples of such methods may be found inAdvanced Bacterial Genetics by R. W. Davis et al., Cold Spring HarborPress, Cold Spring Harbor, N.Y., (1980), which is specificallyincorporated herein by reference. It is preferred, in one embodiment,that the transformation occur at low temperatures, as temperatureregulation is contemplated as a means of regulating gene expressionthrough the use of operational elements as set forth above. In anotherembodiment, if osmolar regulators have been inserted into the vector,regulation of the salt concentrations during the transformation would berequired to insure appropriate control of the synthetic genes.

[0094] If it is contemplated that the recombinant metalloproteinaseinhibitors will ultimately be expressed in yeast, it is preferred thatthe cloning vector first be transferred into Escherichia coli, where thevector would be allowed to replicate and from which the vector would beobtained and purified after amplification. The vector would then betransferred into the yeast for ultimate expression of themetalloproteinase inhibitor.

[0095] The host microorganisms are cultured under conditions appropriatefor the expression of the metalloproteinase inhibitor. These conditionsare generally specific for the host organism, and are readily determinedby one of ordinary skill in the art, in light of the publishedliterature regarding the growth conditions for such organisms, forexample Bergey's Manual of Determinative Bacteriology, 8th Ed., Williams& Wilkins Company, Baltimore, Md., which is specifically incorporatedherein by reference.

[0096] Any conditions necessary for the regulation of the expression ofthe DNA sequence, dependent upon any operational elements inserted intoor present in the vector, would be in effect at the transformation andculturing stages. In one embodiment, the cells are grown to a highdensity in the presence of appropriate regulatory conditions whichinhibit the expression of the DNA sequence. When optimal cell density isapproached, the environmental conditions are altered to thoseappropriate for expression of the portable DNA sequence. It is thuscontemplated that the production of the metalloproteinase inhibitor willoccur in a time span subsequent to the growth of the host cells to nearoptimal density, and that the resultant metalloproteinase inhibitor willbe harvested at some time after the regulatory conditions necessary forits expression were induced.

[0097] In a preferred embodiment of the present invention, therecombinant metalloproteinase inhibitor is purified subsequent toharvesting and prior to assumption of its active structure. Thisembodiment is preferred as the inventors believe that recovery of a highyield of re-folded protein is facilitated if the protein is firstpurified. However, in one preferred, alternate embodiment, themetalloproteinase inhibitor may be allowed re-fold to assume its activestructure prior to purification. In yet another preferred, alternateembodiment, the metalloproteinase inhibitor is caused to assume itsre-folded, active state upon recovery from the culturing medium.

[0098] In certain circumstances, the metalloproteinase inhibitor willassume its proper, active structure upon expression in the hostmicroorganism and transport of the protein through the cell wall ormembrane or into the periplasmic space. This will generally occur if DNAcoding for an appropriate leader sequence has been linked to the DNAcoding for the recombinant protein. The preferred metalloproteinaseinhibitors of the present invention will assume their mature, activeform upon translocation out of the inner cell membrane. The structuresof numerous signal peptides have been published, for example by MarionE. E. Watson in Nuc. Acid Res. 12:515-5164, 1984, specificallyincorporated herein by reference. It is intended that these leadersequences, together with portable DNA, will direct intracellularproduction of a fusion protein which will be transported through thecell membrane and will have the leader sequence portion cleaved uponrelease from the cell.

[0099] In a preferred embodiment, the signal peptide of the E. coli OmpAprotein is used as a leader sequence and is located in a positioncontiguous with the portable DNA sequence coding for themetalloproteinese inhibitor structure.

[0100] Additionally preferred leader sequences include those ofbeta-lactamase, carboxypeptidase G2 and the human signal protein. Theseand other leader sequences are described.

[0101] If the metalloproteinase inhibitor does not assume its proper,active structure, any disulfide bonds which have formed and/or anynoncovalent interactions which have occurred will first be disrupted bydenaturing and reducing agents, for example, guanidinium chloride andβ-mercaptoethanol, before the metalloproteinase inhibitor is allowed toassume its active structure following dilution and oxidation of theseagents under controlled conditions.

[0102] The transcription terminators contemplated herein serve tostabilize the vector. In particular, those sequences as described byGentz et al., in Proc. Natl. Acad. Sci. USA 78: 4936-4940 (1981),specifically incorporated herein by reference, are contemplated for usein the present invention.

[0103] It is to be understood that application of the teachings of thepresent invention to a specific problem or environment will be withinthe capabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and representative processes for their isolation andmanufacture appear in the following examples.

EXAMPLES Example 1 Preparation of Poly(A⁺) RNS from HEF-SA Fibroblasts

[0104] HEF-SA cells were grown to near confluence in 75 cm² T-flasks.Cells were washed twice in Dulbecco's phosphate buffered saline solutionand harvested by the addition of 2 ml of 10 mM Tris, pH 7.5 containing1% w/v SDS (obtained from BDH chemicals, Ltd., Poole, England), 5 mMEDTA and 20 ug/ml protease K (obtained from Boehringer MannheimBiochemicals, Indianapolis, Ind.). Each flask was subsequently washedwith an additional milliliter of this same solution.

[0105] The pooled aliquots from the cell harvest were made to 70 ug/mlin protease K and incubated at 40° C. for 45 minutes. The proteolyzedsolution was brought to a NaCl concentration of 150 mM by the additionof 5 M stock and subsequently extracted with an equal volume ofphenol:chloroform 1:1. The aqueous phase was reextracted with an equalvolume of chloroform. Two volumes of ethanol were added to the aqueousphase and incubated overnight at −20° C. The precipitated nucleic acidswere recovered by centrifugation at 17,500×g for 10 minutes in a BeckmanJ2-21 centrifuge, Beckman Instruments, Palo Alto, Calif., and wereredissolved in 25 ml of 0.1% w/v SDS. This solution was again extractedwith an equal volume of chloroform. The aqueous phase was added to twovolumes of cold ethanol and kept at −20° C. for 2 hours. The precipitatewas collected by centrifugation at 10,000×g for 15 minutes andredissolved in 10 ml of 1 mM Tris, 0.5 mM EDTA, 0.1% SDS, pH 7.5. RNAwas precipitated from this solution by the addition of 10 ml of 4 MLiCl, 20 mM NaoAc, pH 5.0 and incubated at −20° C. for 18 hours. Theprecipitate was again recovered by centrifugation and washed twice with2 M LiCl before redissolving in 1 mM Tris, 0.5 mM EDTA, 0.1% SDS, pH7.5. This solution was stored at −70° C.

Chromatography on Oligo dT Cellulose

[0106] Total cellular RNA prepared as above was ethanol precipitated andredissolved in 0.5 M NaCl. Five ml of RNA at 0.45 mg/ml were applied toa 1 ml column of washed type VII oligo dT cellulose (obtained from PLBiochemicals, Milwaukee, Wis.). The column was then washed with 10 ml of0.5 M NaCl and eluted with 2.0 ml of sterile H₂O. The eluted poly(A⁺)fraction of RNA was ethanol precipitated and dissolved to give a 1 mg/mlsolution in 1 mM Tris, 0.1 mM EDTA, pH 8.0. This was stored at −70° C.

cDNA Synthesis

[0107] Poly(A⁺) RNA was primed with oligo dT (obtained from PLBiochemicals, Milwaukee, Wis.) to serve as a template for cDNA synthesisby AMV reverse transcriptase (obtained from Life Sciences, Inc., St.Petersburg, Fla.). Following the synthesis reaction, the RNA washydrolyzed by the addition of 0.1 volume of 3 N NaOH and incubated at67° C. for 10 minutes. The solution was then neutralized and the cDNApurified by gel filtration chromatography on biogel A 1.5 (obtained fromBioRad Laboratories, Richmond, Calif.) in a 0.7×25 cm column in a 10 mMTris, 5 mM EDTA, and 1% SDS, pH 7.5 solution. Fractions containing cDNAwere pooled and concentrated by ethanol precipitation. The cDNA was dGtailed and purified by gel filtration using the procedure set forthabove. Second strand synthesis was primed with oligo dC and polymerizedin an initial reaction with the large (Klenow) fragment of DNApolymerase (obtained from Boehringer Mannheim). Following second strandsynthesis, E. coli DNA polymerase I (obtained from Boehringer Mannheim)was added and incubation continued to form blunt ends. The doublestranded cDNA was again purified by chromatography. EcoRI restrictionsites within the cDNAs were modified by the action of EcoRI methylase,obtained from New England Biolabs, Beverly, Mass. The cDNA was againpurified and ligated to synthetic EcoRI linkers. Finally, the ends werethen trimmed with the endonuclease and the cDNA purified by gelfiltration. This DNA was ligated into a unique EcoRI site in lambda gt10DNA packaged in vitro and used to infect E. coli strain hf1A accordingto the method set forth by Huynh, T. V., Young, R. A., and Davis, R. W.,in DNA Cloning Techniques, A Practical Approach (ed. Glover, D. M.) (IRLPress oxford), in press, specifically incorporated herein by Preference.Approximately 25,000 recombinants were amplified in this manner.

Screening

[0108] Recombinant-phage-containing sequences of interest were selectedby their preferential hybridization to synthetic oligonucleotidesencoding portions of the primary structure of the desiredmetalloproteinase inhibitor, hereinafter referred to as FIBAC. Theseportions of the protein sequence correspond in part to those set forthin the published literature by Stricklin, G. P. and Welgus, H. G., J.Biol. Chem. 258: 12252-12258 (1983), specifically incorporated herein byreference. Recombinant phage were used to infect E. coli strain hf1A andplated at a density of approximately 2×10³ pfu/150 mm petri dish. Phagewere blotted onto nitrocellulose filters (BA85, Schleicher & SchuellInc., Keene, N.H.), and DNA was denatured and fixed essentially asdescribed by Benton and Davis in Science 196:180-182 (1979) specificallyincorporated herein by reference.

[0109] Using that procedure, the filters were treated sequentially for10-15 minutes each in 0.5 M NaCl, then 10 M Tris, 1.5 M NaCl pH 8.0, andfinally submerged in 2×SSPE. (2×SSPE is 0.36 M NaCl, 20 mM NaH₂PO₄, 2 mMEDTA pH 7.4). Filters were blotted dry and baked 75°-80° for 3-4 hours.Duplicate filters were made of each plate. Filters were prehybridizedfor 1-3 hours at 37° in 5×SSPE containing 0.1×SET, 0.15% NaPPi, and1×Denhardts solutions. Filters were then hybridized for 72 hours at 37°in this same solution containing 5×10⁵ cpm/ml of 5′ end-labeled51-meroligonucleotide specific activity approximately 10⁶ cpm/pmole).Following hybridization, filters were washed six times in 5×SSPEcontaining 0.1×SET and 0.05% sodium pyrophosphate at 37°, then threetimes in 2×SSPE at 21°. These were then blotted dry and autoradiographedon Kodak XAR-5 film at −70° with a Kodak lightening-plus intensifyingscreen. Signals clearly visible from duplicate filters were used to pickphage for plaque purification. Filter preparations and hybridizationprocedures for plaque purification steps were the same as above. Thewashing procedure was simplified to 6 changes of 2×SSPE at 37°. Sixisolates purified by repetitive plating were then arranged on a singlelawn of E. coli strain C600 for testing with subsequent probes.

[0110] Preferential hybridization of the 17-mer to each of the isolates(as opposed to control plaques) was observed under a condition identicalto that used for plaque purification. Probe C was used in a similartest, except that the SSPE concentration during hybridization wasreduced to 4×. Again, each of the isolates demonstrated strongerhybridization to the probe than did control plaques.

Phage Purification and cDNA Characterization

[0111] Quantities of each of the six isolated phage were made by theplate stock technique and purified by serial CsCl block gradientcentrifugation. DNA was extracted from these by dialysis against 50%formamide as described by Davis, R. W., Botstein, D., Roth, J. R., in AManual for Genetic Engineering: Advanced Bacterial Genetics, 1980, ColdSpring Harbor Laboratory, specifically incorporated herein by reference.DNA from each of the isolates was digested with EcoRI and the productswere analyzed by agarose gel electrophoresis. The insert from one of thelarger clones, lambda FIBAC 5, was found to lack internal sites forSalI, HindIII, BamHI, and EcoRI. The cDNA insert was released fromlambda FIBAC 5 DNA and the lambda arms digested by co-digesting withthese four enzymes. The fragments were then ethanol-precipitated andligated into the EcoRI site of plasmid pUC9 without furtherpurification. These plasmids were then used to transform E. coli strainJM83. Transformants were selected on ampicillin containing plates.Plasmids from several transformants were purified and characterized onthe basis of the EcoRI digestion products. One was selected which had aninsert co-migrating with the insert from lambda FIBAC 5 on agarose gelelectrophoresis. This plasmid has been named pUC9-F5/237P10.

Mapping and Subcloning

[0112] The insert in pUC9-F5/237P10 was mapped with respect to internalPstI sites. Double digests with EcoRI and Pst demonstrated threeinternal PstI recognition sites. The entire insert and the componentpieces were subcloned into M13 bacteriophage mp19 and mp18,respectively. Sequencing of the pieces was performed by thedideoxynucleotide method described by Sanger et al. in Sanger, F.,Nicklen, S., and Coulson, A. R., Proc. Natl. Acad.

[0113] Sci. USA 74:5463-5467 (1977), specifically incorporated herein byreference.

[0114] The sequence of the DNA insert from pUC9-F5/237P10 showed an openreading frame which encodes the primary structure of a mature fibroblastcollagenase inhibitor biologically equivalent to that isolable fromhuman skin fibroblasts. The salient features of the sequence are:

[0115] (1) The insert is flanked by EcoRI restriction sites and by G/Cand A/T homopolymeric tracts consistent with the cloning methodology;

[0116] (2) The coding strand is presented in the 5′ to 3′ conventionwith poly C at the 5′ end and poly A at the 3′ end, again consistentwith the techniques employed;

[0117] (3) If the first G in the sequence GTTGTTG immediately adjacentto the 3′ end of the poly C tract is considered as nucleotide 1, then anopen reading frame is presented which encodes the primary structure ofthe mature human fibroblast collagenase inhibitor beginning atnucleotide 34 and continuing through nucleotide 585;

[0118] (4) The termination codon TGA at nucleotides 586 through 588defines the carboxy terminus of the translation product which is thesame as that of the mature protein;

[0119] (5) Nucleotides 1 through 33 define an amino acid sequence whichis not found in the primary structure of the processed protein, butwhich is probably a portion of a leader peptide characteristic ofsecreted proteins;

[0120] (6) The three internal PstI sites have as their first basenucleotides 298, 327, and 448;

[0121] (7) There is a single recognition sequence for the restrictionenzyme TthlllI beginning at nucleotide 78; and

[0122] (8) There is a single recognition sequence for the restrictionendonuclease NcoI beginning at nucleotide 227.

[0123] The sequence of nucleotides 1 through 703 and restriction siteanalysis are shown. FRAG- # SITES FRAGMENTS MENTS ENDS ACC 1 (GTVWAC) 1214 495 (69.8) 214 709 214 (30.2) 1 214 ALU 1 (AGCT) 4 358 358 (50.5) 1358 363 124 (17.5) 482 606 482 119 (16.8) 363 482 606 103 (14.5) 606 7095 (0.7) 358 363 AVA 1 (CQCGPG) 1 536 536 (75.6) 1 536 173 (24.4) 536 709AVA 2 (GGRCC) 3 257 257 (36.2) 1 257 477 220 (31.0) 257 477 572 137(19.3) 572 709 95 (13.4) 477 572 BBV 1 (GCTGC) 1 269 440 (62.1) 269 709269 (37.9) 1 269 BST N1 (CCRGG) 3 344 344 (48.5) 1 344 544 200 (28.2)344 544 557 152 (21.4) 557 709 13 (1.8) 544 557 DDE 1 (CTNAG) 4 186 344(48.5) 365 709 355 186 (26.2) 1 186 360 169 (23.8) 186 355 365 5 (0.7)360 365 5 (0.7) 355 360 ECO R1 (GAATTC) 1 698 698 (98.4) 1 698 11 (1.6)698 709 FNU4H 1 (GCNGC) 2 196 440 (62.1) 269 709 269 196 (27.6) 1 196 73(10.3) 196 269

[0124] FRAG- # SITES FRAGMENTS MENTS ENDS FOK 1 (GGATG) 4 192 274 (38.6)435 709 204 192 (27.1) 1 192 303 132 (18.6) 303 435 435 99 (14.0) 204303 12 (1.7) 192 204 HAE 2 (PGCGCQ) 1 368 368 (51.9) 1 368 341 (48.1)368 709 HAE 3 (GGCC) 3 30 616 (86.9) 93 709 63 33 (4.7) 30 63 93 30(4.2) 63 93 30 (4.2) 1 30 HGI A1 (GRGCRC) 1 552 552 (77.9) 1 552 157(22.1) 552 709 HHA 1 (GCGC) 1 369 369 (52.0) 1 369 340 (48.0) 369 709HINC 2 (GTQPAC) 1 118 591 (83.4) 118 709 118 (16.6) 1 118 HINF 1 (GANTC)2 308 308 (43.4) 1 308 587 279 (39.4) 308 587 122 (17.2) 587 709 HPA 2(CCGG) 4 207 224 (31.6) 372 596 372 207 (29.2) 1 207 596 165 (23.3) 207372 654 58 (8.2) 596 654 55 (7.8) 654 709 HPH 1 (GGTGA) 2 380 380 (53.6)1 380 519 190 (26.8) 519 709 139 (19.6) 380 519

[0125] FRAG- # SITES FRAGMENTS MENTS ENDS MBO 2 (GAAGA) 1 650 650 (91.7)1 650 59 (8.3) 650 709 MNL 1 (CCTC) 5 81 193 (27.2) 81 274 274 174(24.5) 535 709 406 132 (18.6) 274 406 486 81 (11.4) 1 81 535 80 (11.3)406 486 49 (6.9) 486 535 MST 2 (CCTNAGG) 1 185 524 (73.9) 185 709 185(26.1) 1 185 NCI 1 (CCSGG) 2 372 372 (52.5) 1 372 595 223 (31.5) 372 595114 (16.1) 595 709 NCO 1 (CCATGG) 1 227 482 (68.0) 227 709 227 (32.0) 1227 NSP B2 (CVGCWG) 1 197 512 (72.2) 197 709 197 (27.8) 1 197 PST 1(CTGCAG) 3 298 298 (42.0) 1 298 327 261 (36.8) 448 709 448 121 (17.1)327 448 29 (4.1) 298 327 SAU 1 (CCTNAGG) 1 185 524 (73.9) 185 709 185(26.1) 1 185 SAU 3A (GATC) 1 150 559 (78.8) 150 709 150 (21.2) 1 150

[0126] FRAG- # SITES FRAGMENTS MENTS ENDS SAU96 1 (GGNCC) 5 29 220(31.0) 257 477 92 165 (23.3) 92 257 257 137 (19.3) 572 709 477 95 (13.4)477 572 572 63 (8.9) 29 92 29 (4.1) 1 29 SCR F1 (CCNGG) 5 344 344 (48.5)1 344 372 172 (24.3) 372 544 544 114 (16.1) 595 709 557 38 (5.4) 557 595595 28 (3.9) 344 372 13 (1.8) 544 557 SFA N1 (GATGC) 1 193 516 (72.8)193 709 193 (27.2) 1 193 TTH111 1 1 79 630 (88.9) 79 709 (GACNNNGTC) 79(11.1) 1 79

[0127] The following do not appear: AAT 2 AFL 2 AFL 3 AHA 3 APA 1 ASU 2AVA 3 AVR 2 BAL 1 BAM H1 BCL 1 BGL 1 BGL 2 BIN 1 BSSH 1 BST E2 CFR 1 CLA1 ECO R5 FNUD 2 GDI 2 HAE 1 HGA 1 HGI C1 HGI D1 HGI J2 HIND 3 HPA 1 KPN1 MLU 1 MST 1 NAE 1 NAR 1 NDE 1 NRU 1 NSP C1 PVU 1 PVU 2 RRU 1 RSA 1 SAC1 SAC 2 SAL 1 SMA 1 SNA 1 SPH 1 STU 1 TAQ 1 XBA 1 XHO 1 XHO 2 XMA 3 XMN1

[0128]         10         20         30         40         50         60GTTGTTGCTG TGGCTGATAG CCCCAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACAG                              SH                               AA                              UE                               13        70         80         90        100        110        120ACGGCCTTCT GCAATTCCGA CCTCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAGTC  H                T  M           SH                          H  A                T  N           AA                          I  E                H  L           UE                          N  3                1  1           13                          2       130        140        150        160        170        180AACCAGACCA CCTTATACCA GCGTTATGAG ATCAAGATGA CCAAGATGTA TAAAGGGTTC                               S                                A                               U                                A       190        200        210        220        230        240CAAGCCTTAG GGGATGCCGC TGACATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGTC    SD      FS  FN       F  H       A             N    AD      OF  NS       O  P       C             C    UE      KA  UP       K  A       C             O    11      11  12       1  2       1             1        250       260        270        280        290        300 TGCGGATACTTCCACAGGTC CCACAACCGC AGCGAGGAGT TTCTCATTGC TGGAAAACTG                 A            B     M                         P                 V            B     N                         S                 A            V     L                         T                 2            1     1                         1       310        320        330        340        350        360CAGGATGGAC TCTTGCACAT CACTACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAGC  F    H          P                  B           D  A D  O    I          S                  S           D  L D  K    N          T                  T           E  U E  1    1          1                  1           1  1 1        370       380        390        400        410        420 TTAGCTCAGCGCCGGGGCTT CACCAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGTG   AD  HH   N       H                            M   LD  AH   C       P                            N   UE  EA   I       H                            L   11  21   1       1                            1        430        440       450        460        470        480 TTTCCCTGTT TATCCATCCCCTGCAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGAC               F             P                               A               O             S                               V               K             T                               A               1             1                               2       490        500        510        520        530        540CAGCTCCTCC AAGGCTCTGA AAAGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCGG A   M                                   H                 MA L   N                                   P                 NV U   L                                   H                 LA 1   1                                   1                 11        550       560        570        580        590        600 GAGCCAGGGCTGTGCACCTG GCAGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGGA   B        H    B                A               H        NH   S        G    S                V               I        CP   T        I    T                A               N        IA   1        1    1                2               1        12        610       620        630        640        650        660 GTGGAAGCTGAAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGACA     A                                               M    H     L                                               B    P     U                                               O    A     1                                               2    2        670       680        690        700 ATGAAATAAA GAGTTACCAC CCAGCAAAAAAAAAAAGGAA TTC                                         E                                        C                                        O                                        1

Example 2 Expression of Collagenese Inhibitor in E. coli

[0129] In this Example, a preferred method of coupling a preferredportable DNA sequence to the 5′ end of the cloned cDNA is set forth.This involves making a nucleolytic cleavage at a specified point withinthe coding sequence and reconstructing the desired portion of the codingsequence by means of synthetic oligonucleotides in a manner that allowsits excision and recombination (i.e., by incorporating usefulrestriction sites).

[0130] Trimming the 5′ end of the coding region will be accomplished bysynthesizing both strands of the DNA extending from the TthlllI site inthe 5′ direction and ending in a BamHI overhang. This syntheticoligonucleotide, referred to as FIBAC A, has the following features:

[0131] (1) Codon selection has been biased toward those most frequentlyfound in the genes of highly expressed bacterial proteins;

[0132] (2) A methionine codon from which to initiate translation hasbeen provided immediately upstream from the cysteine which begins thecoding region of human processed FIBAC;

[0133] (3) The spacing of the BamHI site to the methionine codon is suchthat when cloned into pUC8, the coding region of FIBAC will be in-framewith the 5′ end of the beta-galactosidase gene;

[0134] (4) An in-frame stop codon and Shine Dalgarno sequence are alsopresented. Translation of this frame for the amino terminal portion ofthe beta-galactosidase is terminated at the TAA codon, and translationof FIBAC should be initiated at the following ATG;

[0135] (5) Codons have been selected to create a HgiAI site beginningwith the G in the FIBAC initiation codon; and

[0136] (6) There is a PvuI site separated by one base from the 3′ end ofthe BamHI sequence.

[0137] The structure of FIBAC A is GA TCC GCG ATC GGA GTG TAA GAA ATGTGC ACT      G CGC TAG CCT CAC ATT CTT TAC ACG TGA TGC GTT CCG CCG CATCCG CAG ACT GCT TTC ACG CAA GGC GGC GTA GGC GTC TGA CGA AAG TGC AAC TCTGAG C ACG TTG AGA CTG GA

[0138] FIBAC A is synthesized using the ABI DNA synthesizer (FosterCity, Calif.) as a series of four component oligonucleotides. Componentoligonucleotide FA1 is: GATCC GCGAT CGGAG TGTAA GAAAT GTGCA CTTGCComponent oligonucleotide FA2 is: GGAACG CAAGT GCACA TTTCT TACAC TCCGATCGCG Component oligonucleotide FA3 is: GTTC CGCCG CATCC GCAGA CTGCTTTCTG CAACT CTGAC C Component oligonucleotide FA4 is: AGGTC AGAGT TGCAGAAAGC AGTCT GCGGA TGCGG C

[0139] The remainder of the coding portion of the FIBAC gene is isolatedas the 3′ TthlllI to EcoRI fragment generated by a double digest ofpUC9-F5/237P10 with these enzymes.

[0140] A synthetic linker is made to couple the 3′ end of the TthlllI toEcoRI fragment to a SalI site. These oligonucleotide will be designed torecreate the SalI site and destroy the EcoRI site. The linker iscomprised of the oligonucleotides linker A1 and linker A2.

[0141] Linker A1 is: AATTGGCAG

[0142] Linker A2 is: TCGACTGCC

[0143] These oligonucleotides and oligonucleotides FA1-FA4 are kinasedseparately and annealed in equal molar ratios with the TthlllI to EcoRI3′ end of the cDNA and BamHI/SalI cut mp19RF DNA. The ligated DNA isused to transfect JM105. Plaques are picked by their color in thepresence of IPTG and X-gal and by hybridization to oligonucleotide FA2.Several positive plaques are to be sequenced. Those containing thedesigned sequence are subcloned into BamHI/SalI digested pUC8.Translation of the FIBAC gene in this construct is coupled totranslation initiated for beta-galactosidase. This expression vector isreferred to a pUC8-Fic.

[0144] Coupling translation of FIBAC to translation initiated for otherhighly expressed proteins is similarly arranged. For example, a portionof the OmpA gene which contains the Shine-Dalgarno and initiatormethionine sequences has been synthesized. This sequence encodes theentire signal peptide of OmpA protein and had convenient restrictionsites, including those for EcoRI, EcoRV, PvuI, and StuI.

[0145] The sequence of the sense strand is:        10         20         30         40         50         60GAATTCGATA TCTCGTTGGA GATATTCATG ACGTATTTTG GATGATAACG AGGCGCAAAA E   TE                                   F          M   H C   AC                                   O          N   H O   QO                                   K          L   A 1   15                                   1          1   1        70         80         90        100        110 AATGAAAAAGACAGCTATCG CGATCGCAGT GGCACTGGCT GGTTTCGCTA CCGTA             A    NF  PS              L    RN  VA             U    UU  UU              1    12  1A   120        130 GCGCAGGCCTCTGGT AAAAGCTT H   S H M          HA H   T A N          IL A   U EL          NU 1   1 3 1          31

[0146] This sequence is hereinafter referred to as OmpA leader. Couplingthe translation of FIBAC to OmpA is accomplished by cutting the pUC8-Ficwith PvuI and SalI and isolating the coding region. This, together withthe EcoRI to PvuI fragment isolated from OmpA leader, will be clonedinto EcoRI/SalI-cut pUC8. As ir the prior example, transcription isdriven by the lac promoter and regulated by the lac I gene product atthe lac operator. This FIBAC expression vector is referred to aspUC8-F/OmpAic.

[0147] To effect the translocation of FIBAC out of the inner cellmembrane, an appropriate leader sequence is added to the amino terminusof FIBAC. The protein thus produced will be translocated and processedto yield the mature form.

[0148] To effect such a translocation, a FIBAC gene encoding thesignal-peptide of the E. coli OmpA protein continuous with thestructural region of FIBAC is created. This particular FIBAC genenecessitates having in frame stop codons at the 5′ end of the FIBACcoding region changed. To accomplish this, the portion of the 5′ codingregion from pUC8-Fic that extends from the HgiA: site to the NcoI siteis isolated. Upstream sequences are resynthesized as a linker havingcohesive ends from BamHI and HgiAI and containing an internal StuI site.This is synthesized as two oligonucleotides, linker B1 and linker B2.

[0149] Linker B1 is: GATCCCAGGCCTGCA

[0150] Linker B2 is: GGCCTGG

[0151] Linkers B1 and B2 are kinased separately and annealed in equalmolar ratios with the HgiAI to NcoI fragment described above andBamHI/NcoI cut pUC8-Fic. The resulting construct has the coding sequenceof FIBAC in frame with the translation of th amino terminus ofbeta-galactosidase. Translation of this sequence forms a fusion proteinwith FIBAC. This plasmid is referred to as pUC8-Ff.

[0152] Attaching the OmpA leader sequences to the coding region of FIBACis accomplished by ligating EcoRI/Stul cut pUC8-Ff with ar excess of thepurified EcoRl to StuI fragment of OmpA leader. Followingtransformation, plasmids from several colonies will be characterized byhybridization. Those that have incorporated t OmpA leader fragment arecharacterized further to verify the structure. This plasmid, pUC8-FOmpAl, will direct the synthesis of a fusion protein beginning in thesignal peptide of the E. coli OmpA protein and ending in human FIBAC.The signals present in the OmpA portion of the protein effect theprotein's export from the cytoplasm and appropriate cleavage from theprimary structure of FIBAC.

[0153] If the efficiency of expression were to be compromised by thesequence of the leader peptide or its combination with FIBAC either atthe protein or at the nucleic acid level, the gene could be altered toencode any of several known E. coli leader sequences.

[0154] Transcription of all of the genes discussed is effected by thelac promoter. As in the case of initiation sites for translation, thepromoter and operator region of the gene may be interchanged. FIBAC mayalso be expressed from vectors incorporating the lambda P_(L) promoterand operator (O_(L)), and the hybrid promoter operator, Tac as describedin Amann, E., Brosius, J., and Ptashne, M. Gene 25:167-178 (1983),specifically incorporated herein by reference. Excision of thoseportions of the gene including ribosome binding site structural regionand 3′ nontranslated sequences and insertion in alternate vectorscontaining the P_(L) or Tac promoter makes use of the unique restrictionsites that flank these structures in pUC8-F/OmpAic and pUC8-F/OmpAl.Insertion of the EcoRI to SalI fragment from either into similarlydigested plasmid pDP8 effects transcription of these genes directed bythe lambda P_(L) promoter. Transcriptional regulation would betemperature sensitive by merit of the cI857 mutation harbored on thissame plasmid.

[0155] Putting similar gene fragments into the transcription unit of theTac promoter will be accomplished by first isolating the EcoRV to SalIfragment. This, together with the synthetic Tac promoter sequence whichis flanked by BamHI and PvuII sites and which contains the lac operatorwill be inserted into the BamHI to SalI sites of pBR322 or preferablyderivatives. The derivatives in this case refer to constructs containingeither the lacI gene or the I^(q) gene.

[0156] Expression of FIBAC in host microorganisms other than Escherichiais considered. Yeast and bacteria of the genera Bacillus, Pseudomonas,and Clostridium may each offer particular advantages. The processesoutlined above could easily be adapted to others.

[0157] In general, expression vectors for any microorganism will embodyfeatures analogous to those which we have incorporated in the abovementioned vectors of E. coli. In some cases, it will be possible tosimply move the specific gene constructs discussed above directly into avector compatible with the new host. In others, it may be necessary ordesirable to alter certain operational or structural elements of thegene.

Example 3

[0158] The human collagenase inhibitor may be readily purified afterexpression in a variety of microbes. In each case, the spectrum ofcontaminant proteins will differ. Thus, appropriate purification stepswill be selected from a variety of steps already known to give a goodseparation of the human collagenase inhibitor from other proteins andfrom other procedures which are likely to work.

[0159] If the inhibitor is not secreted from the microbes, it may forminclusion bodies inside the recombinant microbes. These bodies areseparated from other proteins by differential centrifugation afterdisruption of the cells with a French Press. The insoluble inclusionbodies are solubilized in 6 M guanidine hydrochloride or 8 M urea, andthe inhibitor protein is more completely solubilized by reaction of itscysteines with sodium sulfite. At any time subsequent to this step, thecysteines are converted back to their reduced form with dithiothreitol.Once the inhibitor protein is solubilized from inclusion bodies,immunoaffinity chromatography using antibodies raised against theunfolded inhibitor are used for purification before refolding.

[0160] The inhibitor can be refolded according to the protocol mentionedin Example 6, infra. After refolding of the inhibitor, or if theinhibitor is secreted from the microbes, purification from otherproteins is accomplished by a variety of methods. Initial steps includeultrafiltration through a 50 K dalton cutoff membrane or ammoniumsulfate fractionation. Other useful methods include, but are not limitedto, ion-exchange chromatography, gel filtration, heparin-sepharosechromatography, reversed-phase chromatography, or zinc-chelatechromatography. All of these steps have been successfully used inpurification protocols. Additional high resolution steps includehydrophobic interaction chromatography or immunoaffinity chromatography.After purification, the metalloproteinase inhibitor is preferably atleast 90-95% pure.

Example 4 Purification of Human Collagenase Inhibitor from HumanAmniotic Fluid

[0161] Human amniotic fluid obtained from discarded amniocentesissamples was pooled and 6 liters were subjected to ultrafiltrationthrough a 100 kD MW cutoff filter, obtained from Millipore Corporation,in a Millipore Pellicon Cassette System. The eluate was concentratedthrough a 10 kD cutoff filter, obtained from Millipore Corporation, thenthrough an Amicon PM-10 membrane. Aliquots (10 ml) of concentratedamniotic fluid were eluted through a 2.5×100 cm column of UltrogelAcA54, obtained from LKB Corporation, which was equilibrated with pH7.6, 0.05 M hepes, 1 M sodium chloride, 0.01 M calcium chloride, and0.02% sodium azide (all chemicals were obtained from Sigma ChemicalCompany). Fractions containing the inhibitor were collected and pooled,dialyzed against pH 7.5, 0.025 M Hepes buffer containing 0.01 M calciumchloride and 0.02% sodium azide, and loaded onto a 1.5×28 cmheparin-sepharose CL-6B (obtained from Pharmacia, Inc.) columnequilibrated with the same buffer. This column was rinsed with 1 literof the above buffer and eluted with a linear gradient of 0-0.3 M sodiumchloride. The fractions from the largest peak of inhibitor activity,eluting at about 0.1-0.15 M sodium chloride, were pooled, concentratedto 1 ml, and loaded onto a Synchropak rp-8 reverse phase HPLC columnequilibrated with 0.05% trifluoroacetic acid (Aldrich Chemical Company).The column was eluted with a linear gradient of 0-40% acetonitrile (J.T. Baker Chemical Company) at ½% per minute. All fractions wereimmediately dried in a Savant speed-vac concentrator to removeacetonitrile, and redissolved in pH 7.5, 0.1 M Hepes before assay. Theinhibitor eluted between 32-38% acetonitrile. Fractions containing theinhibitor were pooled, and 100 ul aliquots were eluted over a Bio-radbiosil-TSK 250 HPLC gel filtration column. The pooled peaks of inhibitoractivity contained 0.1 mg of inhibitor, which was over 95% pure asjudged by SDS- polyacrylamide gel electrophoresis.

Example 5 Purification of Human Fibroblast Collagenase Inhibitor fromHuman Embryonic Skin Fibroblast Serum-Free Medium

[0162] Human embryonic skin fibroblasts were grown in serum-free tissueculture medium. Ten liters of this medium were collected, dialyzedagainst pH 7.5, 0.02 M hepes buffer containing 0.02% sodium azide and0.01 M calcium chloride, and applied to a 2.8×48 cm column ofheparin-sepharose CL-6B (Pharmacia, Inc.) equilibrated with the samebuffer. The column was rinsed with 2 liters of this buffer and was theneluted with linear gradient of 0-0.3 M sodium chloride contained in thisbuffer. The fractions obtained were tested for the presence of inhibitorby their ability to inhibit human fibroblast collagenase. The fractionscorresponding to the peak of activity were those obtained near 0.15 Msodium chloride. These fractions were concentrated to about 5 ml byultrafiltration through an Amicon YM10 filter and the concentrate wasapplied in four separate runs to a 250×4.1 mm Synchropak rp-8 reversephase HPLC column, equilibrated with 1% trifluoroacetic acid. The columnwas eluted with a 0-60% linear gradient of acetonitrile in 0.1%trifluoroacetic acid. The gradient was run at ½% acetontrile per minute.The inhibitor eluted in two sharp peaks between 26-29% acetonitrile. Allfractions were immediately dried in a Savant speed-vac concentrator,redissolved in pH 7.5, 0.1 M Hepes, and assayed. At least 1.2 mg ofcollagenase inhibitor was recovered, which was 90-95% pure. Thismaterial gives a single band when run on a 17.5% reducing SDS gel. Aftercarboxymethylation of the cysteines and elution through the same rp-8column under identical conditions, the inhibitor is suitably homogenousfor protein sequencing.

Example 6

[0163] It is contemplated that the human collagenase inhibitor can bereadily refolded into its native structure from its denatured stateafter expression of its gene in a microbe and separation of thecollagenase inhibitor from most of the other proteins produced by themicrobe. By analogy to the conditions necessary for the refolding ofother disulfide-containing proteins as set forth by Freedman, R. B. andHillson, D. A., in “Formation of Disulfide Bonds,” In: The Enzymology ofPost-Translational Modification of Proteins, Vol. 1, R. B. Freedman andH. C. Hawkins, eds., pp. 158-207 (1980), specifically incorporatedherein by reference, refolding of the human collagenase inhibitor shouldoccur in solutions with a pH of 8.0 or greater. At this pH, thecysteines of the protein are partially ionized, and this condition isnecessary for the attainment of native disulfide bond pairings. Theinhibitor concentration should be relatively low, less than 0.1 mg/ml,to minimize the formation of intermolecular disulfide-linked aggregateswhich will interfere with the refolding process.

[0164] Since the stability of the refolded (native) disulfide bondedstructure relative to the unfolded (reduced) structure depends on boththe solution oxidation-reduction potential and the concentrations ofother redox-active molecules, it is contemplated that the redoxpotential should be buffered with a redox buffer giving a potentialequivalent to a reduced: oxidized glutathione ratio of 10. The preferredconcentration range of reduced glutathione would be 0.1-1.0 mM. Athigher concentrations, mixed disulfides will form with protein, reducingthe yield of the refolded (native) structure. The relative stabilitiesof the unfolded protein and the native structure, and thus the rate andyield of refolding, will also depend on other solution variables, suchas the pH, temperature, type of hydrogen-ion buffer, ionic strength, andthe presence or absence of particular anions or cations as discussed inPrivalov, P. L., “Stability of Proteins, Small Globular Proteins,” inAdvances in Protein Chemistry, Vol. 33, pp. 167-236, (1979),specifically incorporated herein by reference. These conditions vary forevery protein and can be determined experimentally. It is contemplatedthat addition of any molecule that strongly prefers to bind the native(as opposed to the unfolded) structure, and which can be readilyseparated afterwards from the native (refolded) protein, will increasenot only the yield but the rate of re-folding. These molecules includemonoclonal antibodies raised against the native structure, and otherproteins which tightly bind the native collagenase inhibitor, such asthe mammalian enzymes collagenase or gelatinase

Example 7

[0165] The second preferred sequence as set forth herein, i.e.,````````10`````````20`````````30`````````40`````````50`````````6GGCCATCGCC GCAGATCCAG CGCCCAGAGA GACACCAGAG AACCCACCAT GGCCCCCTTH``````F`````XB````HH`````````````````````````````N````SA``````N`````HI````AH`````````````````````````````C````AE``````U`````ON````EA`````````````````````````````O````U3``````1`````21````21`````````````````````````````1````1````````70`````````80`````````90````````100````````110````````12GACCCCTGGC TTCTGCATCC TGTTGTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCAC````B``````````SF````````````````````````````````````S H````S``````````FO````````````````````````````````````A A````T``````````AK````````````````````````````````````U E````1``````````11````````````````````````````````````1 3```````130````````140````````150````````160````````170````````18TGTGTCCCAC CCCACCCACA GACGGCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCAA`````````````````````````H````````````````T``M```````````SH`````````````````````````A````````````````T``N```````````AA`````````````````````````E````````````````H``L```````````UE`````````````````````````3````````````````1``1```````````13```````190````````200````````210````````220````````230````````24TTCGTGGGGA CACCAGAAGT CAACCAGACC ACCTTATACC AGCGTTATGA GATCAAGAT```````````````````H```````````````````````````````````S```````````````````I```````````````````````````````````A```````````````````N```````````````````````````````````U```````````````````2```````````````````````````````````A```````250````````260````````270````````280````````290````````300ACCAAGATGT ATAAAGGGTT CCAAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCTAC```````````````````````````SD``````FS``FN```````F``H```````A```````````````````````````AD``````OF``NS```````O``P```````C```````````````````````````UE``````KA``UP```````K``A```````C```````````````````````````11``````11``12```````1``2```````1```````310````````320````````330````````340````````350````````360ACCCCCGCCA TGGAGAGTGT CTGCGGATAC TTCCACAGGT CCCACAACCG CAGCGAGGAG```````N````````````````````````````````A````````````B`````M```````C````````````````````````````````V````````````B`````N```````O````````````````````````````````A````````````V`````L```````1````````````````````````````````2````````````1`````1```````370````````380````````390````````400````````410````````420TTTCTCATTG CTGGAAAACT GCAGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGTC```````````````````P`````F````H````````````````````P```````````````````S`````O````I````````````````````S```````````````````T`````K````N````````````````````T```````````````````1`````1````1````````````````````1 ```````430GCTCCCTGGA AC ````B ````S ````T ````1

[0166] has the following restriction sites: FRAG- # SITES FRAGMENTSMENTS ENDS ACC 1 (GTVWAC) 1 295 295 (68.3) 1 295 137 (31.7) 295 432 AVA2 (GGRCC) 1 338 338 (78.2) 1 338 94 (21.8) 338 432 BBV 1 (GCTGC) 1 350350 (81.0) 1 350 82 (19.0) 350 432 BIN 1 (GGATC) 1 14 418 (96.8) 14 43214 (3.2) 1 14 BST N1 (CCRGG) 2 65 360 (83.3) 65 425 425 65 (15.0) 1 65 7(1.6) 425 432 DDE 1 (CTNAG) 1 267 267 (61.8) 1 267 165 (38.2) 267 432FNU4H 1 (GCNGC) 3 8 269 (62.3) 8 277 277 82 (19.0) 350 432 350 73 (16.9)277 350 8 (1.9) 1 8 FOK 1 (GGATG) 4 76 197 (45.6) 76 273 273 99 (22.9)285 384 285 76 (17.6) 1 76 384 48 (11.1) 384 432 12 (2.8) 273 285 HAE 2(PGCGCQ) 1 19 413 (95.6) 19 432 19 (4.4) 1 19 HAE 3 (GGCC) 5 1 258(59.7) 174 432 51 60 (13.9) 51 111 111 50 (11.6) 1 51 144 33 (7.6) 111144 174 30 (6.9) 144 174 1 (0.2) 1 1 HHA 1 (GCGC) 1 20 412 (95.4) 20 43220 (4.6) 1 20 HINC 2 (GTQPAC) 1 199 233 (53.9) 199 432 199 (46.1) 1 199HINF 1 (GANTC) 1 389 389 (90.0) 1 389 43 (10.0) 389 432 HPA 2 (CCGG) 1288 288 (66.7) 1 288 144 (33.3) 288 432 MNL 1 (CCTC) 2 162 193 (44.7)162 355 355 162 (37.5) 1 162 77 (17.8) 355 432 MST 2 (CCTNAGG) 1 266 266(61.6) 1 266 166 (38.4) 266 432 NCO 1 (CCATGG) 2 47 261 (60.4) 47 308308 124 (28.7) 308 432 47 (10.9) 1 47 NSP B2 (CVGCWG) 1 278 278 (64.4) 1278 154 (35.6) 278 432 PST 1 (CTGCAG) 2 379 379 (87.7) 1 379 408 29(6.7) 379 408 24 (5.6) 408 432 SAU 1 (CCTNAGG) 1 266 266 (61.6) 1 266166 (38.4) 266 432 SAU 3A (GATC) 2 14 217 (50.2) 14 231 231 201 (46.5)231 432 14 (3.2) 1 14 SAU96 1 (GGNCC) 4 51 165 (38.2) 173 338 110 94(21.8) 338 432 173 63 (14.6) 110 173 338 59 (13.7) 51 110 51 (11.8) 1 51SCR F1 (CCNGG) 2 65 360 (83.3) 65 425 425 65 (15.0) 1 65 7 (1.6) 425 432SFA N1 (GATGC) 2 75 199 (46.1) 75 274 274 158 (36.6) 274 432 75 (17.4) 175 STY 1 (CCRRGG) 2 47 261 (60.4) 47 308 308 124 (28.7) 308 432 47(10.9) 1 47 TTH111 1 1 160 272 (63.0) 160 432 (GACNNNGTC) 160 (37.0) 1160 XHO 2 (PGATCQ) 1 13 419 (97.0) 13 432 13 (3.0) 1 13

[0167] The following do not appear: AAT 2 AFL 2 AFL 3 AHA 2 AHA 3 ALU 1APA 1 ASU 2 AVA 1 AVA 3 AVR 2 BAL 1 BAM H1 BAN 1 BAN 2 BCL 1 BGL 1 BGL 2BSM 1 BSP 1286 nSSH 1 BST E2 CFR 1 CLA 1 ECO R1 ECO R5 FNUD 2 GDI 2 HAE1 HGA 1 HGI A1 HGI C1 HGI D1 HGI J2 HIND 3 HPA 1 HpH 1 KPN 1 MBO 2 MLU 1MST 1 NAE 1 NAR 1 NCI 1 NDE 1 NHE 1 NOT 1 NRU 1 NSP C1 PVU 1 PVU 2 RRU 1RSA 1 SAC 1 SAC 2 SAL 1 SCA 1 SMA 1 SNA 1 SNA B1 SPE 1 SPH 1 SSP 1 STU 1TAQ 1 XBA 1 XHO 1 XMA 3 XMN 1

[0168] The salient features of this cDNA are:

[0169] 1. The coding strand is presented in the 5′ to 3′ convention withthe polyC tract at the 5′ end.

[0170] 2. If the first G in the sequence GGC CAT CGC CGC is consideredas nucleotide 1, then an open reading frame exists from nucleotide 1through nucleotide 432, which is the 3′ end of this partial cDNA.

[0171] 3. The first methionine in this reading frame is encoded bynucleotides 49 through 51 and represents the initiation site oftranslation.

[0172] 4. The amino acid sequence prescribed by nucleotides 49 through114 is not found in the primary structure of the mature protein, but itis the sequence of the leader peptide of human protein.

[0173] 5. The sequence of nucleotides 82 through 432 is identical to thesequence of nucleotides numbered 1 through 351 in the insert from thefirst preferred sequence of Example 1.

[0174] 6. The amino acid sequence of the mature protein displays twoconsensus sequences for sugar attachment. These sequences, -N-Q-T-prescribed by nucleotides 202 through 210 and -N-R-S- prescribed bynucleotides 346 through 354, are amino acid residues 30 through 32 and78 through 80, respectively, in the mature protein. Both sites areglycosylated in the human inhibitor protein.

[0175] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the processes and productsof the present invention. Thus, it is intended that the presentinvention cover the modifications and variations of this inventionprovided they come within the scope of the appended claims and theirequivalence.

What is claimed is:
 1. A portable DNA sequence comprising a series ofnucleotides capable of directing intracellular production ofmetalloproteinase inhibitors.
 2. The portable DNA sequence of claim 1wherein said sequence is capable of directing intracellular productionof collagenase inhibitors.
 3. The portable DNA sequence of claim 1wherein said nucleotide sequence is:        10         20         30         40         50         6GTTGTTGCTG TGGCTGATAG CCCCAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACA        70         80         90        100        110        12ACGGCCTTCT GCAATTCCGA CCTCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAGT       130        140        150        160        170        18AACCAGACCA CCTTATACCA GCGTTATGAG ATCAAGATGA CCAAGATGTA TAAAGGGTT       190        200        210        220        230        24CAAGCCTTAG GGGATGCCGC TGACATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGT       250        260        270        280        290        30TGCGGATACT TCCACAGGTC CCACAACCGC AGCGAGGAGT TTCTCATTGC TGGAAAACT       310        320        330        340        350        36CAGGATGGAC TCTTGCACAT CACTACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAG       370        380        390        400        410        42TTAGCTCAGC GCCGGGGCTT CACCAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGT       430        440        450        460        470        48TTTCCCTGTT TATCCATCCC CTGCAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGA       490        500        510        520        530        54CAGCTCCTCC AAGGCTCTGA AAAGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCG       550        560        570        580        590        60GAGCCAGGGC TGTGCACCTG GCAGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGG       610        620        630        640        650        660GTGGAAGCTG AAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGACA       670        680        690        700 ATGAAATAAA GAGTTACCACCCAGCAAAAA AAAAAAGGAA TTC


4. The portable DNA sequence of claim 2 wherein said sequence is capableof directing intracellular production of a collagenase inhibitorbiologically equivalent to that isolable from human skin fibroblasts. 5.A recombinant-DNA cloning vector comprising a nucleotide sequencecapable of directing intracellular production of metalloproteinanseinhibitors.
 6. The vector of claim 5 wherein said vector comprises anucleotide sequence containing at least the following nucleotides:        10         20         30         40         50         60GTTGTTGCTG TGGCTGATAG CCCCAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACAG        70         80         90        100        110        120ACGGCCTTCT GCAATTCCGA CCTCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAGTC       130        140        150        160        170        180AACCAGACCA CCTTATACCA GCGTTATGAG ATCAAGATGA CCAAGATGTA TAAAGGGTTC       190        200        210        220        230        240CAAGCCTTAG GGGATGCCGC TGACATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGTC       250        260        270        280        290        300TGCGGATACT TCCACAGGTC CCACAACCGC AGCGAGGAGT TTCTCATTGC TGGAAAACTG       310        320        330        340        350        360CAGGATGGAC TCTTGCACAT CACTACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAGC       370        380        390        400        410        420TTAGCTCAGC GCCGGGGCTT CACCAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGTG       430        440        450        460        470        480TTTCCCTGTT TATCCATCCC CTGCAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGAC       490        500        510        520        530        54CAGCTCCTCC AAGGCTCTGA AAAGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCG       550        560        570        580        590        60GAGCCAGGGC TGTGCACCTG GCAGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGG       610        620        630        640        650        660GTGGAAGCTG AAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGACA       670        680        690        700 ATGAAATAAA GAGTTACCACCCAGCAAAAA AAAAAAGGAA TTC


8. A recombinant-DNA method for microbial production of ametalloproteinase inhibitor comprising: (a) preparation of a portableDNA sequence capable of directing a host microorganism to produce aprotein having metalloproteinase inhibitor activity; (b) cloning theportable DNA sequence into a vector capable of being transferred intoand replicating in a host microorganism, such vector containingoperational elements for the portable DNA sequence; (c) transferring thevector containing the portable DNA sequence and operational elementsinto a host microorganism capable of expressing the metalloproteinaseinhibitor protein; (d) culturing the host microorganism under conditionsappropriate for amplification of the vector and expression of theinhibitor; and (e) in either order: (i) harvesting the inhibitor; and(ii) causing the inhibitor to assume an active, tertiary structurewhereby it possesses metalloproteinase inhibitor activity.
 9. The methodof claim 8 wherein said metalloproteinase inhibitor is collagenaseinhibitor.
 10. The method of claim 8 wherein said portable DNA sequenceis:         10         20         30         40         50         60GTTGTTGCTG TGGCTGATAG CCCCAGCAGG GCCTGCACCT GTGTCCCACC CCACCCACAG        70         80         90        100        110        120ACGGCCTTCT GCAATTCCGA CCTCGTCATC AGGGCCAAGT TCGTGGGGAC ACCAGAAGTC       130        140        150        160        170        180AACCAGACCA CCTTATACCA GCGTTATGAG ATCAAGATGA CCAAGATGTA TAAAGGGTTC       190        200        210        220        230        240CAAGCCTTAG GGGATGCCGC TGACATCCGG TTCGTCTACA CCCCCGCCAT GGAGAGTGTC       250        260        270        280        290        300TGCGGATACT TCCACAGGTC CCACAACCGC AGCGAGGAGT TTCTCATTGC TGGAAAACTG       310        320        330        340        350        360CAGGATGGAC TCTTGCACAT CACTACCTGC AGTTTCGTGG CTCCCTGGAA CAGCCTGAGC       370        380        390        400        410        420TTAGCTCAGC GCCGGGGCTT CACCAAGACC TACACTGTTG GCTGTGAGGA ATGCACAGTG       430        440        450        460        470        480TTTCCCTGTT TATCCATCCC CTGCAAACTG CAGAGTGGCA CTCATTGCTT GTGGACGGAC       490        500        510        520        530        540CAGCTCCTCC AAGGCTCTGA AAAGGGCTTC CAGTCCCGTC ACCTTGCCTG CCTGCCTCGG       550        560        570        580        590        600GAGCCAGGGC TGTGCACCTG GCAGTCCCTG CGGTCCCAGA TAGCCTGAAT CCTGCCCGGA       610        620        630        640        650        660GTGGAAGCTG AAGCCTGCAC AGTGTCCACC CTGTTCCCAC TCCCATCTTT CTTCCGGACA       670        680        690        700 ATGAAATAAA GAGTTACCACCCAGCAAAAA AAAAAAGGAA TTC


11. The method of claim 8 wherein said vector containing said portableDNA sequence is pUC9-F5/237P10.
 12. The method of claim 8 wherein saidhost microorganism is a bacterium.
 13. The method of claim 12 whereinsaid bacterium is a member of the genus Bacillus.
 14. The method ofclaim 13 wherein said bacterium is Bacillus subtilis.
 15. The method ofclaim 12 wherein said bacterium is Escherichia coli.
 16. The method ofclaim 12 wherein said bacterium is a member of the genus Pseudomonas.17. The method of claim 16 wherein said bacterium is Pseudomonasaeruginosa.
 18. The method of claim 8 wherein said host microorganism isa yeast.
 19. The method of claim 8 wherein said yeast is Saccharomycescerevisiae.
 20. The method of claim 8 wherein said inhibitor isharvested prior to being caused to assume said active, tertiarystructure.
 21. The method of claim 8 wherein said inhibitor is caused toassume said active, tertiary structure prior to being harvested.
 22. Ametalloproteinase inhibitor which is biologically equivalent to thecollagenase inhibitor isolable from human skin fibroblasts produced bythe method of claim
 8. 23. The microorganism C600/pUC9-F5/237P10 havingATCC Accession No.
 53003. 24. The portable DNA sequence of claim 1wherein said nucleotide sequence is:        10         20         30         40         50         60GGCCATCGCC GCAGATCCAG CGCCCAGAGA GACACCAGAG AACCCACCAT GGCCCCCTT        70         80         90        100        110        120GACCCCTGGC TTCTGCATCC TGTTGTTGCT GTGGCTGATA GCCCCAGCAG GGCCTGCAC       130        140        150        160        170        180TGTGTCCCAC CCCACCCACA GACGGCCTTC TGCAATTCCG ACCTCGTCAT CAGGGCCAA       190        200        210        220        230        240TTCGTGGGGA CACCAGAAGT CAACCAGACC ACCTTATACC AGCGTTATGA GATCAAGATC       250        260        270        280        290        300ACCAAGATGT ATAAAGGGTT CCAAGCCTTA GGGGATGCCG CTGACATCCG GTTCGTCTAC       310        320        330        340        350        360ACCCCCGCCA TGGAGAGTGT CTGCGGATAC TTCCACAGGT CCCACAACCG CAGCGAGGAC       370        380        390        400        410        420TTTCTCATTG CTGGAAAACT GCAGGATGGA CTCTTGCACA TCACTACCTG CAGTTTCGTC       430        440        450        460        470        480GCTCCCTGGA ACAGCCTGAG CTTAGCTCAG CGCCGGGGCT TCACCAAGAC CTACACTGTT       490        500        510        520        530        540GGCTGTGAGG AATGCACAGT GTTTCCCTGT TTATCCATCC CCTGCAAACT GCAGAGTGGC       550        560        570        580        590        600ACTCATTGCT TGTGGACGGA CCAGCTCCTC CAAGGCTCTG AAAAGGGCTT CCAGTCCCGT       610        620        630        640        650        660CACCTTGCCT GCCTGCCTCG GGAGCCAGGG CTGTGCACCT GGCAGTCCCT GCGGTCCCAG       670        680        690        700        710        720ATAGCCTGAA TCCTGCCCGG AGTGGAAGCT GAAGCCTGCA CAGTGTCCAC CCTGTTCCCA       730        740        750        760        770        780CTCCCATCTT TCTTCCGGAC AATGAAATAA AGAGTTACCA CCCAGCAAAA AAAAAAAGGA